Dyrk1a Phosphorylation of α-Synuclein Mediating Apoptosis of Dopaminergic Neurons in Parkinson's Disease

Objective To investigate the role of aberrant Dyrk1a expression in phosphorylation modification at the α-synuclein serine 129 (Ser129) site to analyze its molecular mechanism in mediating apoptosis of PD. Methods The protein level of P-α-synuclein (Ser129), α-synuclein, Bcl-2, Bax, active caspase 3, GSK3β, PI3K, AKT, and cyclinD1 were detected. The mRNA transcript levels of Dyrk1a and DAT and protein levels of IL-1β, IL-6, COX-2, and TNF-α were detected. Results P-α-synuclein (Ser129), α-synuclein, Bax, active caspase 3, GSK3β, and cyclinD1 expressions were decreased in Dyrk1a-AAV-ShRNA (P < 0.05), and Bcl-2, AKT, and PI3K expressions were increased (P < 0.05). Increased TH protein expression was shown in Dyrk1a-AAV-ShRNA (P < 0.05). Dyrk1a mRNA was decreased in the Dyrk1a-AAV-ShRNA group (P < 0.05), and DAT mRNA was increased (P < 0.05). IL-1β, IL-6, COX-2, and TNF-α protein levels were decreased in Dyrk1al-AAV-Sh-RNA (P < 0.05). Transcriptome sequencing showed that Fam220a, which was expected to activate STAT family protein binding activity and participate in the negative regulation of transcription through RNA polymerase II and protein dephosphorylation showed differentially upregulated expression. The untargeted metabolome showed that the major compounds in the Dyrk1a-AAV-ShRNA group were hormones and transmission mediators and the most metabolism-related pathways. Fam220a showed differentially upregulated expression, and differentially expressed genes were enriched for the neuroactive ligand-receptor interaction, vascular smooth muscle contraction, and melanogenesis-related pathways. Conclusion Abnormal Dyrk1a expression can affect α-synuclein phosphorylation modifications, and dyrk1a knockdown activates the PI3K/AKT pathway and reduces dopaminergic neuron apoptosis. It provides a theoretical basis for the group to further investigate the molecular mechanism.


Introduction
Parkinson's disease (PD) is an age-dependent neurodegenerative disease with a prevalence of approximately 1.7% in people over 65 years of age [1][2][3]. Te pathogenesis of PD remains unclear, and a multiple-hit hypothesis of PD pathogenesis has been proposed, including genetic and environmental factors, and it simultaneously afects neuronal homeostasis, leading to progressive neurodegeneration of dopaminergic neurons [4]. Te degenerative necrosis of dopaminergic neurons in the substantia nigra in PD patients results in the formation of Lewy bodies (LBs) and Lewy neurites (LNs) [5]. LBs and LNs are mainly composed of misfolded a-synuclein, which is one of the key proteins in the pathogenesis of PD. Phosphorylation of the a-synuclein Ser129 site induces an unfolded protein response (UPR) in aa-synuclein, which ultimately leads to functional degenerative necrosis of DA neurons and afects tyrosine hydroxylase (TH) activity [6,7]. Te study of asynuclein phosphorylation mechanisms has become a research hotspot in recent years. Tis result suggests that the phosphorylation level of a-synuclein Ser129 site may have an important correlation with the production of LBS and even dopaminergic neuronal degeneration [8,9].
Te phosphorylation process of Ser129 is dynamic, and its phosphorylated form afects the subcellular assignment of PD-associated mutant genes such as A30P and A53T [10][11][12]. Furthermore, it has been suggested that the binding and dissociation of synaptosomal membranes of mutant asynuclein may be regulated by pathological Ser129 phosphorylation [13]. Te distribution pattern of Dyrk1a in the human brain is specifc to the brain region, cell type, and subcellular compartment. Dyrk1a was nearly identical in the frontal, temporal, and occipital cortices. Immunocytochemistry detected signifcant diferences in the brain structurespecifc and neuron-type-specifc distribution of Dyrk1a, suggesting that the role of Dyrk1a in development, maturation, aging, and degeneration may difer across brain structures and neuronal types [14][15][16]. Dyrk1a has been shown to have multiple biological functions, as refected in its interactions with numerous cytoskeletal, synaptic, and nuclear proteins, including transcription factors and splicing factors [17,18]. Dyrk1a is described as a regulator of a broad spectrum of neurodevelopmental mechanisms that identifes 239 genes deregulated by the overexpression of Dyrk1a through the REST/NRSF chromatin remodeling complex, which suggests a central role for this kinase in brain pathology. Te expression of Dyrk1a in fetal and postnatal neurons, as well as in adult and elderly neuron expression, suggests that the regulation of Dyrk1a is an integral part of neuronal development, maturation, and aging [19]. Dyrk1a plays a key role in neuroproliferation and neurogenesis in the developing brain, and its gene is located on chromosome 21 (21q22.2), a region known as the Down syndrome critical region (DSCR) [20,21]. Due to its location, triplication of the Dyrk1a locus in Down syndrome (DS) results in a 1.5fold increase in Dyrk1a in the fetal and adult brain [22]. In addition, upregulation of Dyrk1a has been reported to promote altered neuronal proliferation in DS patients through specifc phosphorylation of p53 at the Ser15 locus [23].
To date, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are by far the most commonly used neurotoxins to cause a Parkinsonian state by causing severe loss of dopaminergic neurons [24,25]. Te gold standard model for motor symptoms of Parkinson's disease was generated by administering MPTP to the Eastern Hemisphere nonhuman primate (NHP) species [26]. Te MPTP-treated NHP model is most often replicated after systemic injection of MPTP, which readily crosses the bloodbrain barrier and leads to Parkinson's syndrome, manifested by prolonged rest, bradykinesia, postural defcits, and reduced overall movement [27]. MPTP has a broad neurotoxic efect on dopaminergic, adrenergic, and 5-hydroxytryptaminergic neurons outside the substantia nigra. Based on the previous experiments, Dyrk1a gene knockdown phosphorylation modifes a-synuclein and downregulates P-a-synuclein (Ser129) expression. Dyrk1a has direct interaction with Pa-synuclein (Ser129) and GSK-3b, and Dyrk1a gene overexpression inhibits the PI3K/AKT pathway to promote DA neuronal apoptosis. It is suggested that Dyrk1a phosphorylation of a-synuclein mediates apoptosis in dopaminergic neurons. In the study, MPTP-induced PD model mice with tyrosine hydroxylase (TH) as the promoter of adenoassociated virus specifcally silenced the expression of Dyrk1a gene in the midbrain substantia nigra by stereotaxic localization in the striatum of the mouse. It was verifed by behavioral tests, Western blot (WB) technique, immunohistochemistry, immunofuorescence, enzyme-linked immunosorbent assay (ELISA), HE staining, and transmission electron microscopy to further analyze the molecular mechanism of Dyrk1a phosphorylation of a-synucleinmediated apoptosis of dopamine neurons.

Materials and Methods
2.1. Experimental Animals. Sixty 6-8-week-old C57BL/6 male mice weighing 25.20 ± 2.12 g were fed on a 12-hour alternating light/dark cycle at 22.10 ± 1.03°C and 60.12 ± 5.09% relative humidity. Te experiments were started one week after the mice were acclimated to the animal room environment for modeling. Te mice were purchased from the Animal Experiment Center of Xinjiang Medical University, and the experiments were reviewed by the Experimental Animal Ethics Committee of the First Afliated Hospital of Xinjiang Medical University before the start of the experiments, and the experimental operations and handling processes were in accordance with the ethical standards for experimental animals. Control group (n � 15): mice were injected intraperitoneally with 0.9% NaCl, 25 mg/kg, twice a week for fve weeks. MPTP group (model, n � 15): mice were injected intraperitoneally with MPTP, 25 mg/kg, twice a week for fve weeks. Ctrl-AAV-ShRNA (model + MPTP + empty vector, n � 15): empty vector (Ctrl-AAV-ShRNA) was injected locally into the striatum of the midbrain of mice, (coordinates: Bregma AP, −3.0 mm, ML, ±1.3 mm, DV, −4.7 mm; dose: 2.85 × 1012 PFU/mL, 0.5 μL per side). Tree weeks after stereotactic injection, mice were injected intraperitoneally with MPTP, 25 mg/kg, twice a week for fve weeks. Dyrk1a-AAV-ShRNA group (model + MPTP + Dyrk1a-AAV-ShRNA, n � 15): Dyrk1a-AAV-ShRNA was frst injected stereotactically into the nigrostriatal region of the midbrain of mice, (coordinates: Bregma AP, −3.0 mm, ML, ±1.3 mm, DV, −4.7 mm. 3.43 × 1012 PFU/mL, 0.5 μL per side). Tree weeks after stereotactic injection, mice were injected intraperitoneally with MPTP, 25 mg/kg, twice a week for fve weeks.

Behavioral Tests.
Te pole climbing experiment, suspension test, open feld experiment, and grasping force experiment were performed.

Western Blot Experiment and qRT-PCR.
Te protein of each treatment group was extracted. We boiled the upper sample solution in boiling water at 100°C for 5 min to denature the protein and added primary antibody diluted in the blocking solution to the incubation bag and incubated overnight at 4°C. We washed the membrane 5 min × 3 times with TBST and incubated horseradish peroxidase-labeled sheep anti-rabbit secondary antibody (Jackson 1 : 5000) or horseradish peroxidase-labeled sheep anti-mouse secondary antibody (Jackson 1 : 5000) at room temperature for 2 h. We washed the membrane 15 min × 5 times with TBST. Te membrane was made to react with the chemiluminescence detection reagent (Reagent A: Reagent B � 1 :1) for 2 min, the membrane was removed, the excess liquid was shaken of, the PVDF membrane was wrapped with cling flm, and the membrane was developed and fxed in the dark room.
Tissue samples were ground with liquid nitrogen, and total RNA was extracted with Trizol for every 50-100 mg of the tissue to use for qRT-PCR. Total RNA was extracted using Trizol (Invitrogen, USA) and changed into cDNA by the using reverse transcriptase kit (Fermentas, USA). Te cycling conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 10 s, 50-60°C for 35 s, and 72°C for 30 s. Temperature increases were 72°C for 5 min. Te RT-qPCR analysis was performed with the Light Cycler 480 RT-qPCR System (Roche, Basel, Switzerland). Fold changes in gene expression were estimated using the CT comparative method, normalizing GAPDH. CT values relative to control samples are as follows: ΔCt � Ct (assayed samples)-Ct(β-actin); ΔΔCt � ΔCt − ΔCt control; and fold diference � 2 −(ΔΔCT) .

HE Staining.
Te tissue blocks were taken, fxed, conventionally parafn-embedded, cut into 4 μm sections, dewaxed in xylene, washed by all levels of ethanol to water, stained with hematoxylin for 5 min, and rinsed with tap water. Hydrochloric acid ethanol fractionation was performed for 30 s along with tap water immersion for 15 min or warm water (about 50°C) for 5 min. Te tissue sections were subjected to a series of steps for preparation. First, the sections were treated with eosin solution and left for 2 minutes. Ten, conventional dehydration was performed, followed by transparency treatment. Te sections were sealed afterwards. Finally, slices were obtained using an orthomosaic microscope, with feld of view magnifcations of ×10, ×100, ×200, and ×400, respectively.

Immunohistochemical Staining.
Immunohistochemical assay (IHC)-baked slices: put in a 68°C incubator about 20 min, tissue rehydration, 3% H2O2 room temperature for 10 min, washed with PBS 3 times, 5 min each time to remove endogenous enzymes, trypsin and dilution solution diluted at 1 : 3 and added dropwise to the tissue, and incubated at 37°C for 10 min. We diluted the primary antibody with the prepared closure solution. Te primary antibody (1 : 200 dilution) was incubated overnight at 4°C in the refrigerator. We added the HRP-labeled secondary antibody and incubated for 30 min at 37°C and removed the tissue sections from the 37°C incubator, washed 3 times with PBS for 5 min each time, and absorbed the liquid around the tissue with an absorbent paper. 50 μL of concentrated DBH was mixed with 1 mL of DAB substrate, added dropwise on the tissue, observed the brown shade under the microscope, and rinsed with tap water when the color reaches the optimum. Te tissue slices underwent hematoxylin staining, followed by dehydration using a gradient of alcohol. Subsequently, the slices were sealed.
2.6. ELISA. Te plates were sealed tightly with sealing flm and incubated at 37°C for 30 minutes. We diluted the 30-fold concentrated washing solution 30-fold with distilled water and set aside and discarded the sealing membrane, shook of the liquid, flled with washing solution, and repeated this 5 times and pat dry. Te enzyme reagent was added 50 μl/per well, except for blank wells. We warmed up, washed, and added 50 μl of the chromogenic agent A and then 50 μl of chromogenic agent B to each well, shook and mixed gently, and developed for 15 minutes at 37°C and avoided light. Ten, we added 50 μl of termination solution per well to terminate the reaction.

Transmission Electron Microscopy.
Te material was taken and fxed with 1% osmium acid prepared in 0.1 M phosphate bufer PB (PH7.4) for 2 h at room temperature avoiding light, rinsed 3 times in 0.1 M phosphate bufer PB (PH7.4), dehydrated at room temperature, osmotically embedded, ultrathin sections, copper mesh stained in 2% uranyl acetate saturated alcohol solution avoiding light for 8 min; washed 3 times in 70% alcohol; washed 3 times in ultrapure water; 2.6% lead citrate solution avoiding carbon dioxide staining for 8 min; washed with ultra-pure water 3 times; and used a flter paper to slightly blot-dry. We observed the material under a transmission electron microscope and collected images for analysis.

Metabolomics Analysis.
Te sample was precisely measured and extracted, and then the supernatant metabolites were extracted by centrifugation for liquid-liquid mass spectrometry (LC-MS). Te chromatographic conditions were as follows: the column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Milford, USA), mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), and the injection volume was 2 μL. Te column temperature was 40°C. Te metabolomics software Progenesis QI (WatersCorporation, Milford, USA) was used for peak extraction, alignment, and identifcation, and the fnal data matrix contained the retention time, peak area, and mass-to-charge ratio. Te information was obtained for postprocessing and raw letter analysis. Diferential metabolites were performed. Based on hypergeometric analysis, KEGG enrichment analysis was performed for diferential metabolites.

Transcriptome Sequencing.
Total RNA was extracted, and the concentration, purity, and RIN were examined using Nanodrop2000 and Agilent2100. Te Illumina TruseqTM RNA sample prep kit was used for library construction. Using magnetic beads with oligo (dT), mRNA can be isolated from total RNA. Te mRNA can be broken randomly, and small fragments of about 300 bp can be separated by magnetic beads. Six-base random primers are added to synthesize one-stranded cDNA, followed by two-stranded synthesis to form a stable double-stranded structure. Te end repair mix is added to make it fat-terminated, followed by the addition of an "A" base at the 3′ end to join the Y-junction. Sequencing was performed based on Illumina Novaseq 6000. Diferential gene analysis was performed using the diferential analysis software DESeq2, and the diferential gene screening criteria were |log2FC| ≥ 1 and p value <0.05. Based on hypergeometric analysis, functional enrichment analysis of GO and KEGG was performed for diferential genes and p < 0.05 was considered as the signifcant term.
2.10. Statistical Analysis. SPSS 22.0 was used for data processing, and GraphPad Prism 9.00 software was used for analysis and graphing. Te experimental data were expressed as the mean ± standard deviation, and the data diferences between the two groups were analyzed by Student's t-test, the variance discrepancy was tested by the tʹ test, the comparison between multiple groups was analyzed by oneway analysis of variance (ANOVA), and the LSD was used to analyze between the two groups. * : p < 0.05, * * : p < 0.01, * * * : p < 0.001 vs. the control group; #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs. the MPTP group; the diferences were statistically signifcant.

Phenotype Analysis.
Te average pulling force value was 82.69 ± 15.84 g for the control, 62.07 ± 13.94 g for the model, 63.33 ± 11.03 g for Ctrl-AAV-ShRNA, and 74.09 ± 13.87 g for Dyrk1a-AAV-ShRNA. Te model group and the Ctrl-AAV-ShRNA group had lower pulling force (P < 0.001) (Figure 1(a)), suggesting that Dyrk1a knockdown could increase the grasping force of the limb of mice. Te time spent in the control group was 6.1 ± 1.732, the model was 10.57 ± 2.839, Ctrl-AAV-ShRNA was 11.18 ± 2.117, and Dyrk1a-AAV-ShRNA was 5.587 ± 1.71. Te diference was statistically signifcant (P < 0.001) in the MPTP + Dyrk1al-AAV-ShRNA group (Figure 1(b)). Te suspension time was 178.7 ± 36.5 in the control, 42.63 ± 12.51 in the model, 41.7 ± 35.87 in Ctrl-AAV-ShRNA, and 167.9 ± 38.59 in Dyrk1a-AAV-ShRNA. Te differences in MPTP + Dyrk1al-AAV-ShRNA were statistically signifcant (P < 0.001) (Figure 1(c)). Compared with the model group, the total distance of movement was signifcantly increased in MPTP + Dyrk1al-AAV-Sh-RNA (P < 0.05) (Figure 2(a)). Te latency within the frst frame was signifcantly higher in the MPTP + Dyrk1al-AAV-Sh-RNA group (P < 0.05) (Figure 2(b)). Te dwell time in the central region of the MPTP + Dyrk1al-AAV-Sh-RNA group was signifcantly lower (P < 0.01) (Figure 2(c)). Te results of the number of standing observations showed that the number of standing observations was signifcantly increased in MPTP + Dyr-k1al-AAV-Sh-RNA (P < 0.05) (Figure 2(d)). We observed the activity of mice in the central area in each group and found that the model and Ctrl-AAV-ShRNA were less likely to stay or traverse in the central area and MPTP + Dyrk1al-AAV-ShRNA was more active and traversed the central area (P < 0.01) (Figure 2(e)).

Pathological
Analysis. Te brain tissue of Ctrl-AAV-Sh-RNA showed that vacuolation, neuronal atrophy, individual infammatory cells, and neuronal degeneration were improved in MPTP + Dyrk1a-AAV-ShRNA and no infammatory cells were present (Figure 3(a)). TH was positively expressed in MPTP + Dyrk1a-AAV-ShRNA (Figure 3(b)). In the Ctrl-AAV-ShRNA group, neuronal cytosolic organelles were reduced, mitochondria were swollen and deformed, vacuoles appeared in the cytoplasm, the nucleus was obviously wrinkled, and the nucleolus density was increased (Figure 3(c)). In the MPTP + Dyrk1al-AAV-Sh-RNA group, nigrostriatal neurons were cytosolic. Te damaged neurons entered the recovery period, the nucleus morphology of the neurons was close to normal, the mitochondrial morphology was improved than before, and the number of increased coarse endoplasmic reticulum was gradually restored (Figure 3(c)).
Functional annotation analysis of GO and KEGG was performed for the diferential genes (Figure 7(a)). Diferentially expressed genes in Ctrl-AAV-ShRNA has mainly cellular process, biological regulation, response to stimulus, developmental process, and metabolic process in the biological process (Figure 7(b)). Diferential genes of Ctrl-AAV-ShRNA were mainly enriched in antigen processing and presentation, complement and coagulation cascades, and protein digestion and absorption pathways in the organism system. In environmental information processing were cell adhesion molecules, cytokine-cytokine receptor interaction, and viral protein interaction with cytokine and cytokine receptors. Metabolism-related pathways were enriched for arachidonic acid metabolism, thiamine metabolism, primary bile acid biosynthesis, retinol metabolism, and nitrogen metabolism pathways. (Figure 7(c)).
Te MPTP + Dyrk1a-AAV-ShRNA group had 228 differentially expressed genes, including 131 upregulated and 97 downregulated genes. Te diferentially upregulated expression genes in Dyrk1a-AAV-ShRNA were Fam220a, which was expected to activate STAT family protein binding activity and to be involved in negative transcriptional regulation through RNA polymerase II and protein dephosphorylation. Diferentially downregulated expression gene was Tmem215, which maintained endothelial cell survival to promote angiogenesis. Diferentially expressed genes in Dyrk1a-AAV-ShRNA had mainly cellular process, biological regulation, developmental process, and multicellular organismal process. Te diferential genes in Dyrk1a-AAV-ShRNA were enriched for the neuroactive ligand-receptor interaction, vascular smooth muscle contraction, and melanogenesis-related pathways (Figure 7(c)).

Discussion
PD is a progressive neurodegenerative disorder that afects approximately 1% of the population over 55 years of age, with the highest prevalence in people aged 85 years and older [28]. Te clinical manifestations of PD are due to extensive loss of dopaminergic neurons in the nigrostriatal pathway and neuronal dysfunction in the dopaminergic, 5hydroxytryptaminergic, adrenergic, and cholinergic neurotransmitter systems [29]. Both metabolites and proteins refect the physiological and pathological state of the individual. Analysis of these data types may help identify sensitive and efective markers for early disease detection [30]. Andrea et al. performed ultrahigh performance liquid chromatography mass spectrometry analysis of plasma from 21 patients with PD and suggested that tyramine could be used as a marker for early PD and suggested that tyramine, noradrenaline, and tyrosine could be used as prognostic markers [31,32]. Wichter et al. performed a highperformance liquid chromatography study of monoamines in the plasma of patients with PD and found a signifcantly higher homovanillic acid/dopamine ratio and a lower 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio, emphasizing the involvement of multiple neurotransmitter systems in the disease [33,34]. Sonnien et al. observed increased production of LRRK2 mutant synaptophysin by astrocytes from α-mutant PD patients, altered metabolism and calcium homeostasis, increased cytokine release, increased levels of polyamines and their precursors, and decreased levels of lysophosphatidylethanolamine [35]. Tese fndings suggest that astrocytes may be involved in the pathogenesis of PD.
Understanding the altered metabolic pathways and metabolites involved in the development and progression of the disease can help better understand the underlying relevant biological alterations. In this study, brain tissue samples from a mouse model of MPTP-induced chronic Parkinson's disease were metabolomically characterized by an ultra-performance liquid chromatography mass spectrometry untargeted metabolome. Compared with the control group, the diferential metabolic pathways in Ctrl-AAV-ShRNA were metabolism-related pathways, in which the metabolic pathways of cofactors and vitamins, amino acid metabolism, other amino acid metabolism, and carbohydrate metabolism-related pathways were dominant. Te major compounds in the Dyrk1a-AAV-ShRNA group were hormones and transmission mediators. Te Dyrk1a-AAV-Sh-RNA group had the most metabolism-related pathways, among which the metabolic pathways of cofactors and vitamins and carbohydrate metabolismrelated pathways were dominant. In this experiment, brain tissue samples were sequenced by transcriptome sequencing. Te diferentially upregulated expression genes in the Dyrk1a-AAV-ShRNA group were Fam220a, which were expected to activate STATfamily protein binding activity and were expected to be involved in negative transcriptional regulation through RNA polymerase II and protein dephosphorylation. Diferential downregulated expression of gene Tmem215, which maintains endothelial cell survival to promote angiogenesis. Te results of KEGG pathway analysis showed that the diferential genes in the model were mainly enriched in human disease-related pathways, which were basal cell carcinoma, Cushing's syndrome,      Figure 7: Te volcano and enrichment analysis of GO and KEGG for diferential genes.

Conclusion
In PD model mice with low specifc knockdown of DAergic neurons in the midbrain Dyrk1a gene, P-α-synuclein (Ser129), α-synuclein, Bax, active caspase 3, GSK3β, and cyclinD1 expressions were decreased in Dyrk1a-AAV-ShRNA and Bcl-2, AKT, and PI3K expressions were increased. IL-1β, IL-6, COX-2, and TNF-α protein levels were decreased. Te untargeted metabolome showed that the major compounds in the Dyrk1a-AAV-ShRNA group were hormones and transmission mediators and the most metabolism-related pathways. Transcriptome sequencing showed that Fam220a, which was expected to activate STAT family protein binding activity and participate in negative regulation of transcription, showed diferentially upregulated expression. Diferentially expressed genes were enriched for the neuroactive ligand-receptor interaction, vascular smooth muscle contraction, and melanogenesisrelated pathways. Abnormal Dyrk1a expression can afect α-synuclein phosphorylation modifcations, and dyrk1a knockdown activates the PI3K/AKT pathway and reduces dopaminergic neuron apoptosis. Tis study required further functional validation experiments and verifcation of its clinical signifcance, and further analysis was also needed.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest.