Lymphocyte antigen 96: A new potential biomarker and immune target in Parkinson's disease

Background: Lymphocyte antigen 96 ( LY96 ) plays an important role in innate immunity and has been reported to be associated with various neurological diseases. However, its role in Parkinson's disease (PD) remains unclear. Methods: Transcriptome data from a total of 49 patients with PD and 34 healthy controls were downloaded from the Gene Expression Omnibus (GEO) database to analyse the expression pattern of LY96 and its relationship with gene function and immune-related markers. In addition, peripheral blood samples were collected from clinical patients to validate LY96 mRNA expression levels. Finally, an in vitro cell model of PD based on highly differentiated SH-SY5Y cells was constructed, with small interfering RNA-silenced LY96 expression, and LY96 mRNA level, cell viability, flow cytometry, and mitochondrial membrane potential assays were performed. Results: The results of the analyses of the GEO database and clinical samples revealed significantly abnormally high LY96 expression in patients with PD compared with healthy controls. The results of cell experiments showed that inhibiting LY96 expression alleviated adverse cellular effects by increasing cell viability, reducing apoptosis, and reducing oxidative stress. Gene set enrichment analysis showed that LY96 was positively correlated with T1 helper cells, T2 helper cells, neutrophils, natural killer T cells, myeloid-derived suppressor cells, macrophages, and activated CD4 cells, and may participate in PD through natural killer cell-mediated cytotoxicity pathways and extracellular matrix receptor interaction pathways. Conclusion: These findings suggested that LY96 might be a novel potential biomarker for PD, and offer insights into its immunoregulatory role.


Introduction
Parkinson's disease (PD) is a common degenerative disease of the central nervous system, affecting an estimated 1.2 % of people over >65 years of age worldwide (Vijiaratnam et al., 2021).Its pathological characteristics are the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies, gradually leading to irreversible motor dysfunction, which imposes a heavy burden on patients, families, and society (Ascherio and Schwarzschild, 2016;Jankovic and Tan, 2020).The exact factors of PD are currently not clear, although age, Abbreviations: ΔΨm, mitochondrial transmembrane potential; AUC, area under the curve; BP, biological processes; CC, cellular components; CCK-8, cell counting kit-8; CD44, cluster of differentiation 44; CCR2, C-C chemokine receptor type 2; DMEM, Dulbecco's Modified Eagle Medium; ECM, extracellular matrix; Em, emission wavelength; Ex, excitation wavelength; GAPDH, glyceraldehyde phosphate dehydrogenase; GEO, Gene Expression Omnibus; GO, Gene Ontology; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; HMGB, high mobility group box; IC50, half inhibitory concentration; KEGG, Kyoto Encyclopedia of Genes and Genomes; LA, Lymphocyte antigen; LBP, lipopolysaccharide binding protein; LY96, Lymphocyte antigen 96; MD2, myeloid differentiation 2; MDSCs, myeloid-derived suppressor cells; MF, molecular function; MMP, mitochondrial membrane potential; MPP + , N-methyl-4-phenylpyridinium iodide; MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; PBS, phosphate-buffered saline; PD, Parkinson's disease; PPI, protein-protein interaction; ROC, receiver operating characteristic; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA; ssGSEA, single-sample gene set enrichment analysis; TLR, toll-like receptor; TIRAP, TIR domain containing adaptor protein; UPDRS, Unified Parkinson's Disease Rating Scale.genetic susceptibility, oxidative stress, and environmental exposure are all associated with the onset of PD (Gao et al., 2020).Genetic factors play an important role in the occurrence and development of PD, with an estimated 5-10 % of PD cases can be attributed to familial genetic mutations (Zhang et al., 2018).In recent decades, approximately 100 different genes or loci have been found to be associated with susceptibility to PD (Subramaniyan et al., 2023).However, this cannot fully summarize the vast genetic landscape of PD, and precise and effective molecular targets for PD diagnosis and treatment are lacking (Saad and Saad, 2023).Therefore, the in-depth study of the genetic aetiology of PD, and the discovery of new diagnostic biomarkers have profound clinical significance.
Many independent studies have shown that the enhancement and persistence of immune response may be a driving factor for neuronal death in PD (Marogianni et al., 2020).Lymphocyte antigen (LA) molecules play an important role in antigen presentation to T cells, and an increasing evidence suggests that LA is closely related to the occurrence of neurodegenerative diseases (Hobson and Sulzer, 2022).LA-mediated T cells may interact in an antigen-dependent or independent manner to shape the inflammatory cascade that occurs in neurodegenerative diseases, causing neuronal death and central nervous system damage (Augusto and Hollenbach, 2022).Lymphocyte Antigen 96 (LY96), a member of the LA family, plays an important role in innate immunity (Li et al., 2023a).LY96 is closely related to neurodegenerative diseases.For example, high expression of LY96 increases oxidative stress and neuroinflammation in Alzheimer's disease (AD), making it a potential target ligand for AD treatment (Eslami et al., 2019).Fang ZP et al. reported LY96 association with the immune response in ischaemic stroke (Fang et al., 2021), while Petrone AB et al. proposed LY96 as an immune biomarker for the diagnosis of mild traumatic brain injury (Petrone et al., 2017).However, the role of LY96 in the pathological process of PD remains unknown.
Therefore, this study investigated the correlation between LY96 and PD pathogenesis, as well as the changes in the inflammatory environment of PD.This study is the first to demonstrate abnormally high LY96 expression in PD through our analysis of 49 PD cases from four public datasets.Additionally, data from clinical samples and cell experiments further support our evidence of LY96's involvement in the pathological process of PD.Further functional analysis and exploration revealed the profound impacts of LY96 on the immune environment of patients with PD, indicating that LY96 may be a potential immune marker for PD.Ultimately, our discoveries expand our knowledge of the molecular function of LY96 and provide new potential biomarkers and data for PD, particularly in the field of immune regulation (Fig. 1).

Data collection
Four datasets (GSE7621, GSE20141, GSE49036, and GSE20163) were downloaded from the Gene Expression Omnibus (GEO; htt p://www.ncbi.nlm.nih.gov/geo), a public database containing highthroughput gene expression data submitted by researchers worldwide.The experimental dataset comprised the GSE7621, GSE20141, and Fig. 1.The flowchart of this study.
H. Peng et al.GSE49036 datasets, which include brain tissue data from samples from 41 patients with PD and 25 healthy controls.The validation dataset (GSE20163) contains data from 8 patients with PD and 9 healthy controls.Detailed information on these datasets is provided in Supplementary Table 1.The R package "boxplot" was used to determine the expression levels of characteristic genes in the PD and control groups.Receiver operating characteristic (ROC) analysis was performed using the "pROC" package in R to determine the diagnostic accuracy of PDcharacteristic genes.

Clinical samples
After receiving approval from the Ethics Committee of the Henan Provincial People's Hospital (approval number: 2023087), clinical samples were selected from the inpatient population of Henan Provincial People's Hospital between July and December 2023.According to the diagnostic criteria of PD based on the British PD Society Brain Bank, two neurologists conducted diagnostic evaluations on hospitalized patients with PD.The confirmed patients were then evaluated using the Unified Parkinson's Disease Rating Scale (UPDRS) and clinical staging (Hoehn and Yahr [H-Y] rating).In this study, the selection criteria for patients in the disease group were as follows: (a) UPDRS scale score of 20-50; (b) H-Y staging between stages 2-4; (c) PD course >5 years; (d) no other neurodegenerative diseases or chronic diseases (hypertension, diabetes).The healthy control group has also confirmed to have no history of neurological or chronic diseases, and no other limiting factors.Peripheral blood samples were collected from these participants on an empty stomach in the morning, and detailed information on the clinical samples used in this study is provided in Supplementary Table 2.
Small interfering RNA (siRNA) molecules against LY96 (si-LY96) were synthesised by GenePharma Biotechnology Co. Ltd. (Shanghai, China).Lipofectamine 2000 (Thermo Fisher Scientific, USA) was used at a 3.5:1 transfection reagent-to-duplex RNA ratio for transfection in an Opti-MEM medium.SH-SY5Y cells in the logarithmic growth phase were treated with LY96 siRNA.After 24 h of incubation, fresh medium was added to the dishes, and the cells were cultured for another 24 h.To ensure the efficiency of gene suppression, four different sequences of siRNA were selected for siRNA design (Table 1).RNA was extracted for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection 24 h after transfection.

RT-qPCR
RNA in clinical blood samples is efficiently extracted through RNA simple Total RNA Kit (Adsorption column type; TIANGEN, BeiJing, China), using whole blood samples without the need for centrifugation.RNA from highly differentiated SH-SY5Y cells was extracted using Trizol reagent (TIANGEN, Beijing, China).Collect extracted total RNA and a reverse transcription kit (MCE, Shanghai, China) was used to produce single-stranded cDNA, and a one-step qRT-PCR kit (MCE, Shanghai, China) was used for RT-qPCR to measure LY96 expression.The 2 − ΔΔCt technique was used to evaluate fold-changes in mRNA expression levels using glyceraldehyde phosphate dehydrogenase (GAPDH) as the internal reference for standardisation of LY96 expression levels.All primer sequences used in this study are provided in Table 2.

Cell counting kit-8 (CCK-8) assay
Cell viability was measured using a CCK-8 kit (MCE, Shanghai, China) according to the manufacturer's instructions.SH-SY5Y cells were inoculated into 96-well plates at 5 × 10 3 cells per 100 μL.Next, 10 μL of CCK-8 reagent was added to each well and the plates were incubated at 37 • C for 24 h, with CCK-8 solution in cell-free medium and untreated cell medium as the blank and control wells, respectively.The absorbance of each well was measured at 450 nm using a microplate reader.

Flow cytometry
The apoptosis rate was determined 48 h after transfection using a membrane-associated protein V propidium iodide (PI) cell doublestaining apoptosis detection kit (Vazyme, Nanjing, China).After centrifugation, washing, and resuspending the cells, 5 uL of PI and 5 uL of membrane-associated protein V-FITC (Vazyme, Nanjing, China) were added to the SH-SY5Y cells and they were incubated in the dark at room temperature for 10 min.Next, 400 uL of binding buffer was added to each tube and mixed.The flow cytometry (Beckman, USA) parameters were: excitation wavelength (Ex) = 488 nm, emission wavelength (Em) = 530 nm.Surviving, early apoptotic, late apoptotic, and necrotic cells were counted.

Mitochondrial membrane potential assays
A JC-1 detection kit (MCE, Shanghai, China) was used to determine mitochondrial membrane potential (MMP, ΔΨM).SH-SY5Y cells were inoculated at 5 × 10 4 cells per well in a six-well plate and incubated at 37 • C for 24 h.The cells were then rinsed with phosphate-buffered saline (PBS) and cold water and incubated with JC-1 (1 μg/mL) in the dark for 20 min.Finally, the cells were rinsed with PBS and imaged using a fluorescence microscope (Olympus, Japan) at an excitation wavelength of 488 nm.

Table 1
The sequence of si-LY96 in our study.

Table 2
The primer sequences of GAPDH and LY96 in this study.

Sample source Primer Name
Sequence (5′-3′) Peripheral whole blood specimen Peng et al. function (MF), cellular components (CC), and biological processes (BP) with which genes are associated.Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was also performed to reveal functional signalling pathways involving PD genes.GO and KEGG enrichment analyses were performed using the R (version 4.0.3)"clus-terProfiler" and "DOSE" packages, respectively.Additionally, a proteinprotein interaction (PPI) network with overlapping candidate genes was constructed using the STRING database (https://cn.string-db.org/).Cytoscape software was used to identify hub genes in the PPI network.Finally, the function of the hub genes was further explored using the "clusterProfiler" and "enrichplot" packages in R. Based on the Pearson correlation between each hub gene and other genes, a single-gene set enrichment analysis (GSEA) was performed using an ordered gene expression matrix.

Correlation analysis of the immune environment
Gene set variation analysis (GSVA) was performed using the "GSVA" package in R. ssGSEA was performed based on the expression profiles of PD and control samples to estimate the infiltration levels of immune cells and immune functions.The "pheatmap" package in R was used to visualise heatmaps of 29 immune cells and immune functions.Correlation heatmaps were generated between the risk scores and 16 immune cell and 13 immune function scores using the "Corrplot" package in R. Spearman's correlation analysis was performed to reveal the correlations between candidate central genes and immune cells, as well as immune function.

Statistical analysis
The statistical results of RT-qPCR, CCK8 experiment, cell apoptosis investigation, and MMP of SH-SY5Y cells were analyzed using One-Way analysis of variance on GraphPad Prism software, Version 8.4.3.The Unpaired t-test in on was used to analyse the expression of LY96 in clinical samples.Pearson analysis was used to screen the co-expressed genes of LY96 in R software.P < .05 was considered statistically significant, and all experiments in vitro were repeated three times.

Significantly increased LY96 expression in the transcriptome of PD
Three experimental datasets (GSE7621, GSE20141, and GSE49036) were analyzed to explore LY96 expression in this study.A composite matrix expression profile column containing 21,605 genes was generated after standardising the gene expression profile.The heatmap showed increased LY96 expression in patients with PD (Fig. 2A).Subsequently, according to the boxplot, single-gene expression analysis showed significant LY96 overexpression in patients with PD (P < .001,Fig. 2B).In the ROC curve analysis, the area under the curve (AUC) value was 0.772, indicating that LY96 has good diagnostic value for distinguishing patients with PD from healthy individuals (Fig. 2C).The repeatability analysis based on the validation dataset (GSE20163) also showed increased LY96 expression in patients with PD (P < .01,Fig. 2D).The results of ROC analysis confirmed the diagnostic accuracy of LY96 in PD (AUC = 0.715, Fig. 2E).Finally, the result of RT-qPCR experiment from clinical peripheral blood samples showed significantly higher LY96 mRNA expression in patients with clinical PD than that in healthy individuals (P < .01)(Fig. 2F).Taken together, these results demonstrated abnormally high LY96 expression in patients with PD compared with healthy individuals, implying that LY96 might play a role in the pathological progression of PD.

The successfully established PD model is the basis for in vitro experimental validation
To explore the potential relationship between LY96 and the pathological progression of PD, highly differentiated SH-SY5Y cells were used for in vitro experimental validation (Xicoy et al., 2017;Xie et al., 2010), and a PD disease model was constructed with MPP+ (Langston, 2017).Firstly, normally growing cells were exposed to different concentrations of MPP + (0, 100, 250, 500, 600, 750, 1000, and 2000 umol/L).The H. Peng et al. absorbance values of the cells were measured after 24 h.The CCK8 assay results showed that MPP + reduced cell viability in a dose-dependent manner (Fig. 3A), and the half inhibitory concentration (IC50) of the cells was 538.3 uM (Fig. 3B).For the convenience of the experiment and based on previous literature, 500 uM was used as the experimental concentration of MPP + in the subsequent experiments.
siRNA was used to inhibit LY96 expression.To ensure the inhibition efficiency, four different sequences of siRNA were constructed for cell transfection.The RT-qPCR results revealed that si-LY96#2 had the most significant inhibitory effect (Fig. 3C).Thus, this sequence was used in the subsequent experiments.

High LY96 expression affects normal cellular physiological activity in PD and can serve as a risk factor for PD
To investigate the effect of LY96 on cellular life activities in PD, four groups were established for cell experiments: blank control, MPP + , MPP + -siRNA negative control (MPP + -siNC), and MPP + -siLY96.The RT-qPCR results verified the successful establishment of these experimental groups (Fig. 4A).In SH-SY5Y cells, the neurite protrusion growth of MPP + -induced cells was obviously shortened compared with that of normal cells, while cells with inhibited LY96 expression showed cell protrusions with morphology approaching that of healthy cells (Fig. 4B,  C).The results of the CCK-8 assay showed that LY96 inhibition improved the MPP + -induced decrease in SH-SY5Y cell viability and that this upregulatory effect on cell viability remained stable over time (Fig. 4D).In addition, MPP + significantly promoted SH-SY5Y cell apoptosis, an effect that was dampened after LY96 inhibition (Fig. 4E, F).As shown in Fig. 5A, the red fluorescence of healthy control cells was more pronounced, whereas green fluorescence was more pronounced in cells treated with MPP + .LY96-silenced cells showed a higher number of JC-1 aggregates than that in other cells treated with MPP + (both P < .0001,Fig. 5B).In summary, high LY96 expression led to a trend of abnormal cell growth and also affected normal cellular activities such as proliferation, apoptosis, and organelle function.

LY96 is involved in the synthesis or transport process of dopamine and co-gene expression analysis in PD
To explore the biological functions and potential mechanistic pathways of LY96 in PD, multiple functional enrichment analyses were conducted.GO analysis to analyse the biology of the differentially expressed genes, including BP, CC, and MF revealed enrichment of 108, 37, and 28 pathways in each category, respectively.The results of GO analysis showed that the genes mainly affected synaptic transmission (dopamine synthesis, exocytosis, and neuronal migration), neuronal synapses (axons, dendrites, and actin cytoskeleton), and chemical ion transmembrane transmission in PD (Fig. 6A).Among them, LY96 mainly participated in the dopamine biosynthetic process (Fig. 6B).Subsequently, the results of KEGG analysis indicated that genes in the PD dataset were mainly related to metabolic pathways, neuroactive ligandreceptor interactions, cAMP signalling pathways, MAPK signalling pathways, and alcoholism (Fig. 6C).LY96 mainly clustered in dopaminergic synapses and metabolic pathways (Fig. 6D).Finally, the PPI network showed the top 10 genes with the highest functional relevance LY96, including lipopolysaccharide binding protein (LBP), Toll-like receptor (TLR)1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MYD88), cluster of differentiation 44 (CD44), high mobility group box 1 (HMGB1), CD14, LY86, and TIR domain containing adaptor protein (TIRAP) (Fig. 6E).Specific analysis was performed on the gene correlation (Fig. 6F).In summary, LY96 participates in the synthesis or transport of dopamine process via individual or co-gene interactions, thus affecting the physiological activity of dopaminergic neurons in PD.

LY96 affects the immune environment disorder of PD, especially T lymphocytes
To explore the impact of LY96 on the immune environment of PD, the results of immuneCor-analysis revealed the expression differences of 28 common immune cells between patients with PD and healthy controls (Fig. 7A).Subsequently, the ssGSEA results showed that LY96 was positively correlated with T1 helper cells, T2 helper cells, neutrophils, natural killer T cells, myeloid-derived suppressor cells (MDSCs), macrophages, eosinophils, and activated CD4+ cells (all P < .05,Fig. 7B).Furthermore, LY96 expression was significantly correlated with macrophage expression (R = 0.4, P < .001,Fig. 7C).Among T lymphocytes, LY96 was positively correlated with T1 helper cells, regulatory T cells, and activated CD4+ T cells (Fig. 7D-F).LY96 was also highly correlated with eosinophils (R = 0.22, P < .001,Fig. 7G) and MDSC (R = 0.3, P < .001,Fig. 7H) in PD.In short, the differences in the expression levels of immune cells suggest that LY96 may disrupt the immune environment in PD, which is related to the pathological dysregulation of PD immunity.

LY96 participates in the immunopathological changes of PD through multiple immune regulatory pathways
GSEA performed to explore the underlying mechanisms of LY96 and immune environment changes in PD revealed that LY96 enriched six immune-related pathways in PD (Fig. 8A), including the well-known natural killer cell-mediated cytotoxicity, extracellular matrix (ECM) receptor interaction, apoptosis, neuroactive ligand-receptor interaction, basal cell carcinoma, and alanine aspartate and glutamate metabolism pathways (Fig. 8B-G).Meanwhile, after gene set clustering, GSVA analysis revealed 23 expression mechanisms related to LY96, including multiple positively correlated metabolic pathways and negatively H. Peng et al. correlated NOTCH signalling pathways (Fig. 8H).Combined, the GSEA and GSVA results indicated that LY96 might be involved in the immune regulatory pathways of PD, including NK cell-mediated cytotoxicity pathways, and play a potential role in the immunopathology of PD.

Discussion
LA molecules not only regulate antigen presentation process but also affect T cell activation, participate in inflammatory cascade reactions, and are associated with neuronal death and central nervous system damage in neurodegenerative diseases (Aliseychik et al., 2018).As LY96 retains the functions of the LY family, recent researche has focused on revealing its role in various neurological diseases.Eslami et al. reported that LY96 is association with the immune response in AD and is a potential target ligands for AD treatments (Eslami et al., 2019).In addition, LY96 may enhance cognitive impairment by inducing neuroinflammation in sepsis-related encephalopathy (Li et al., 2023b).However, the role of LY96 in the pathological process of PD has been rarely reported.Current evidence suggests that LY96 is a risk factor for central nervous system diseases, motivating us to explore the potential association between LY96 and PD.
This study investigated the potential connections between LY96 expression and the pathological progression of PD.Firstly, analysis of the GEO database revealed abnormally high LY96 expression in patients with PD compared with normal healthy individuals, while the ROC curve analysis confirms its meaningful diagnostic value.Subsequently, H. Peng et al. the RT-qPCR results of clinical haematological specimens indicated that LY96 was also significantly overexpressed in patients with PD.This different perspective of these analyses increased the credibility of our findings.Previous studies have demonstrated that risk genes are highly expressed and promote PD pathogenesis (Nalls et al., 2019).For example, parkin RBR E3 ubiquitin protein ligase (PRKN1) is highly expressed in PD tissues and is associated with poor patients prognosis (Zhang et al., 2018).These findings are similar to our findings in the early stages.Therefore, we hypothesize that high LY96 expression might be a risk factor for PD.
To reinforce our hypothesis that LY96 may be a pathogenic gene in PD development and progression, we constructed an in vitro experimental model of PD.The results of the cell experiments showed that inhibiting LY96 expression improved cell viability and apoptosis levels, leading to the development of cellular activities towards normal morphology.The abnormal proliferation and apoptosis of nerve cells are critical processes in the degenerative changes of the nervous system, which may be related to the initial pathological changes of neurodegenerative diseases such as PD (Tönges et al., 2018).Moreover, we found that LY96 can cause functional impairments in mitochondria.Recent studies have also shown that mitochondrial dysfunction plays an important role in the development and destructive consequences of PD (Moradi Vastegani et al., 2023).On the one hand, mitochondria are the center of energy metabolism in organisms (van der Bliek et al., 2017).Dysfunction of mitochondria can lead to abnormal metabolism of cell proteins and lipids, excessive production of intracellular reactive oxygen species, and cause cell death (Annesley and Fisher, 2019).On the other hand, mitochondrial damage can affect cellular autophagy function, as damaged proteins and organelles accumulate within the cell, leading to cell necrosis and neuronal degeneration and degeneration (Harrington et al., 2023).Therefore, our findings provide data support for the possibility that LY96 may be a potential risk gene for PD, and the abnormally high LY96 expression may inhibit the normal physiological activity of nerve cells, which is involved in pathological changes in patients with PD.
Next, we investigated the mechanisms by which LY96 exerted pathological changes in PD.The results of the GO analysis suggested that LY96 is involved in dopamine biosynthesis.The interaction of LY96 with proteins within the dopaminergic synapse, as revealed by KEGG pathway aggregation, suggests a more complex regulatory role that could affect synaptic transmission and neuronal signalling pathways in PD.Moreover, the progressive reduction of dopaminergic neurons is a pathological feature in PD, and the integrity and synaptic transmission of dopaminergic neurons play an important role in PD (Surmeier, 2018).Furthermore, LY96 does not act alone.We observed the co-expression of LY96 with TLRs and MYD88.TLRs and MYD88 are key components of innate immunity involved in immune response, inflammation, and oxidative stress processes (Zandieh et al., 2023;Zhang et al., 2009).The co-expression of LY96 and other immune-related genes may link neuroinflammation with neurodegeneration, providing broader evidence for their functional expression.Furthermore, the presence of LY96 in metabolic pathways underscores the metabolic disturbances observed in PD, which may be integral to disease progression (Cirstea et al., 2020;Verugina et al., 2020;Li et al., 2023c).In short, LY96 may play an important role in the malignant progression of PD, including dopamine synthesis and transport pathways, metabolism disorders, and coexpression of immune genes.
The crosstalk between PD and immune cells drives the dysfunction, shaping the inflammatory cascade reactions that occur in neurodegenerative diseases and causing neuronal death and central nervous system damage (Pajares et al., 2020).Previous studies have shown that high levels of immune cell infiltration in PD can lead to worsening patients' prognosis (Araújo et al., 2022).In the present study, LY96 affected the abnormal expression of immune cells, mainly T cells, NK cells, and macrophages.Increasing evidence suggests a significant increase in T cell (CD8 and CD4) numbers in the substantia nigra of patients with PD when compared with patients without PD, and that T cell responses are associated with the loss of dopaminergic neurones (Williams et al., 2021;Terrabuio et al., 2023).In addition, dysfunction of peripheral monocytes in patients with PD may be accompanied by increased blood- brain barrier permeability induced by chemokines, leading to high invasiveness to the central nervous system (Su et al., 2022;Lauritsen and Romero-Ramos, 2023;Wijeyekoon et al., 2020).Our results are consistent with those of previous studies.Moreover, our in-depth analysis of immune pathways showing the effects of LY96 on the immune environment in PD through NK cell-mediated cytotoxicity and ECM receptor interaction pathways, which was related to pathological deterioration in PD (Menees and Lee, 2022;Downs et al., 2022).Therefore, the association between LY96 and immune environment disorders in PD further strengthens the important role of LY96 in PD pathogenesis.
In summary, to our knowledge, our study is the first to identify high LY96 expression as a potential risk factor for PD.However, this study has a few limitations.Firstly, the sample size was limited and further validation in larger patient cohorts and experimental is required.Secondly, potential inherent biases may have been present during the experiments.
Thirdly, additional functional experiments or epidemiological evidence are needed.Our study can provide data support and ideas for future research, and based on current research results, future researchers should pay attention to the following points.Firstly, it is necessary to study the interaction between LY96 and other neuroinflammatory mediators in the body, a deeper study can be conducted on the relationship between LY96 and the NK-cell pathways.Secondly, future research should appropriately increase the sample size or adopt longitudinal studies to focus on the longitudinal changes of LY96 expression in PD patients.Lastly, future research directions should focus more on clinical translation and application, gene silencing or targeted interference may provide new options for the treatment of PD in the near future.

Fig. 2 .
Fig. 2. The expression pattern of LY96 in PD patients and healthy individuals (A) The results of the heatmap indicated that LY96 expression is increased in PD. (B) Elevated expression of LY96 in PD observed in the experimental dataset (samples from 41 patients with PD and 25 healthy controls).(C) ROC curve demonstrating the diagnostic value of LY96 in PD based on experimental datasets (AUC = 0.740).(D) Elevated expression of LY96 in PD observed in the validation dataset (samples from 8 PD patients and 25 healthy controls).(E) Diagnostic value of LY96 in the validation dataset (AUC = 0.694).(F) Significant upregulation of LY96 expression in PD peripheral blood samples compared to healthy control samples (Including 40 PD patients and 40 healthy individuals).(** P < .01,*** P < .001).

Fig. 5 .
Fig. 5. Assessment of mitochondrial membrane potential using JC-1 fluorescent dye.(A) JC-1 dyes exhibits aggregated red fluorescence in healthy cells, while damaged cells exhibit more green fluorescence.Nuclei were counterstained with DAPI and showing blue fluorescence.(B) Knockdown of LY96 promoted mitochondrial health in MPP + -treated SH-SY5Y cells.(ns p > .05,****p < .0001).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.Functional analysis and gene co-expression analysis of LY96 (A) Circos plot displaying the chromosomal positions of LY96-related genes.The inner circle shows gene density (red peaks), and the outer circle represents the chromosomal locations.(B) Chord diagram illustrating the relationships between LY96-related genes (right) and associated biological processes (left).(C) Bar graph showing the number of genes involved in various signalling pathways related to LY96.(D) Alluvial diagram indicating the flow of genes between pathways and the correlation of each pathway to LY96 expression.(E) Network plot of protein-protein interactions with LY96 at the center.Nodes represent proteins; edges represent interactions.(F) Correlogram showing pairwise correlations between LY96 and related genes.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7 .
Fig. 7. Correlation between immune-related cells and LY96 in PD (A) Heatmap shows the compositions of infiltrated immune cells between healthy control and patients with PD. (B) Heatmap shows the correlation coefficients between LY96 expression and the abundance of different immune cell types.(C -H) Scatter plots illustrating the correlation between LY96 expression (x-axis) and the relative abundance of different immune cells (y-axis), with the Pearson correlation coefficient (R) and P-value indicated.(*P < .05,** P < .01,***P < .001).

Fig. 8 .
Fig. 8. Gene Set Enrichment Analysis (GSEA) of LY96-associated pathways in immune response.(A) Network plot of pathway enrichment based on GSEA and each node represents a pathway.(B-G) Individual enrichment plots for selected pathways.Each plot shows the running enrichment score (green line) and ranks the metric scores (black lines), with red and blue bars representing genes in the gene set and their correlation with LY96 expression.(H) Heatmap summarizing the core enrichment genes across the analyzed pathways with scores indicating the level of correlation to LY96 expression.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)