Integrated analysis of exosomal lncRNA and mRNA expression profiles reveals the involvement of lnc‐MKRN2‐42:1 in the pathogenesis of Parkinson's disease

Abstract Background Parkinson's disease (PD) is a common movement disorder for which diagnosis mainly depends on the medical history and clinical symptoms. Exosomes are now considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material. Long noncoding (lnc) RNA in exosomes plays a critical role in many diseases, including neurodegenerative disease. Aim To study expression differences for lncRNAs in peripheral blood exosomes of PD patients compared with healthy individuals and to look for lncRNAs that might be related to the pathogenesis of PD. Materials and Methods We recruited PD patients along with age‐ and sex‐matched healthy individuals as healthy controls and evaluated levels of lncRNAs extracted from exosomes in plasma samples via next‐generation sequencing and real‐time quantitative PCR. Correlation analysis was conducted for the clinical characteristics of PD patients and the expression of selected lncRNAs. Results We found 15 upregulated and 24 downregulated exosomal lncRNAs in the PD group. According to bioinformatics analyses, we chose lnc‐MKRN2‐42:1 for further study. Interestingly, lnc‐MKRN2‐42:1 was positively correlated with the MDS‐UPDRS III score for PD patients. Conclusion Our study suggested that lnc‐MKRN2‐42:1 may be involved in the occurrence and development of PD.


| INTRODUC TI ON
Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, with median age-standardized annual incidence rates in high-income countries of 14 per 100 000 individuals in the total population, and 160 per 100 000 among those aged 65 years or older. 1 The clinical features of PD include resting tremor, rigidity, bradykinesia, and gesture failure, with a higher risk of nonmotor symptoms, including cognitive impairment and dementia. 2,3 The main pathological changes in PD are the degeneration and death of dopaminergic neurons in the midbrain substantia nigra and significant decreases in dopamine in the striatum, resulting in an imbalance between dopamine and acetylcholine transmitters. The presence of Lewy bodies in substantia nigra cells is considered a pathological marker of PD. However, the exact mechanism underlying the PD remains unclear. 4 Diagnosis of PD mainly depends on the medical history and clinical symptoms, which are easy to misdiagnose. 3 Exosomes are cell-derived membranous structures that originate from the endosomal system. They are present in blood, semen, saliva, urine, amniotic fluid, cerebrospinal, bile, breast milk, and culture medium from cell cultures, and range in size from 30 to 100 nm. 5 Exosomes are now considered an additional mechanism for inter- They also found that cognitive impairment severity and difficulty in performing daily activities were correlated with levels of urinary exosome Ser(P)-1292 LRRK2. 6 Studies have shown that exosomes are involved in the transmission and release of α-synuclein, which is closely related to PD pathogenesis and dysfunction, and is the main component of Lewy bodies. 7,8 Given the difficulty in obtaining brain tissue and the properties of exosomes, it is appropriate to use exosomes rather than brain tissue to study PD.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length greater than 200 nucleotides (nt) that can comprehensively regulate gene expression through a variety of different mechanisms at epigenetic, transcription, posttranscription, and translation levels. 9,10 In 2014, Soreq et al used whole-transcriptome RNA sequencing to screen 13 differentially expressed lncRNAs in peripheral PD leukocytes, five of which contained the spliceosome component U1, which supported the idea that splicing modulations were involved in the disease. Lnc-fr91.3 was believed to be related to muscle rigidity in PD. 11 In 2015, Carrieri et al demonstrated that antisense transcription regulated sense genes functioned at distinct regulatory levels in PD and could be used as a new therapeutic strategy. 12 In 2016, Kraus et al analyzed the lncRNA expression profile of brain tissues from PD patients and normal subjects and found that significant differences in the expression of lncRNA-p21, MALAT1, SNHG1, NEAT1, and H19. Such differences appeared in the early stages of PD and accompanied its development. 13 These studies suggested that lncRNAs play an important role in the pathogenesis of PD, but the expression of lncRNAs in peripheral blood exosomes of PD patients remains unknown. In this study, we screened lncRNAs in peripheral exosomes from PD patients in order to identify valuable lncRNAs for further study.

| Exosome collection
Seven PD patients (Dnr_01 to Dnr_07; age 63 ± 11 years) and seven healthy controls (Dnr_34 to Dnr_40; age 58 ± 9 years) were selected for next-generation sequencing (NGS) of exosome lncR-NAs. There was no significant difference in age between the two groups, and each group consisted of four males and three females.
Peripheral blood samples from individuals were collected in ethylene diamine tetraacetic acid (EDTA) tubes following a regular venipuncture procedure. After centrifugation at 3000 × g for 15 minutes at 4°C, the plasma was aspirated and stored at −80°C before use. The ultracentrifugation method was optimized according to the method previously described. 14 After thawing at 37°C, plasma samples were centrifuged at 3,00 × g for 15 minutes to remove cell debris. The supernatant was then diluted using a sevenfold volume of phosphate-buffered saline (PBS), centrifuged at 13 000 × g for 30 minutes, and processed through a 0.22 μm filter to remove large particles. The supernatant was ultracentrifuged using a P50A72-986 rotor (CP100NX; Hitachi, Brea, CA, USA) at 100 000 × g at 4°C for 2 hours to pellet the exosomes. The pellet was resuspended in PBS and centrifuged again at 100 000 × g at 4°C for 2 hours. After PBS washing, the exosome pellet was resuspended in 100 µL of PBS.

| Exosome identification
Exosomes were identified via TEM, NTA, and Western blotting. A 20-µL aliquot of exosome solution was placed on a copper mesh

| ExoRNA isolation and RNA analyses
Total RNA was extracted and purified from plasma exosomes using a Transcripts longer than 200 nt and having more than two exons were selected as lncRNA candidates and further screened using CPC/CNCI/Pfam, which has the power to distinguish protein-coding from noncoding genes. In addition to the different types of lncRNAs, lincRNA, intronic lncRNA, and antisense lncRNAs were selected using Cuffcompare ( Figure S6).

| GO and KEGG pathway enrichment analysis
We predicted the target genes for the differentially expressed lncR-NAs. Perl script was used to identify adjacent genes in the range of 100kb upstream and downstream of lncRNAs as cis target genes of lncRNAs. LncTar was used to predict the trans target genes of our lncRNAs. 16

| Clinical characteristics and exosome characterization and properties
Clinical characteristics were collected from medical records (Table 1).  Figure 1A), consistent with previous observations. 5 NTA is a method for direct, real-time visualization and analysis of nanoparticles in liquids and relates the rate of Brownian motion to particle size. 20 The mean diameter of exosomes observed via NTA was consistent with the TEM results, confirming that the exosomes were small extracellular vesicles. 21 CD63 and TSG101

Movement Disorder Society-Sponsored Revision
were used as exosome surface protein markers, while calnexin was used as a negative marker, 22 and their presence and absence were verified by our Western blot results ( Figure 1B).  Table S1. The location of genes on chromosomes is significantly related to their function. Therefore, we analyzed the distribution of differentially expressed lncRNA and mRNA sequences on chromosomes ( Figure S2). According to the lncRNA differential  Table S2. ACRBP, CXCL5, ENKUR, and others were highly expressed among PD patients, whereas NME4, CD3D, and ECSCR were highly expressed among healthy subjects.

| Functional annotation and enrichment analysis
We performed functional annotation of the target genes of differentially expressed lncRNAs. Nine cis target genes and seven trans target genes were functionally annotated. GO analysis showed that these genes are involved in "intracellular part," "single-organism cellular process," "heterocyclic compound binding," etc ( Figure 3) The pathways with the most significant enrichment are listed in Figure 3 (B, D). Remarkably, these target genes were enriched in "autophagy," "fatty acid degradation," "pentose phosphate pathway," and "HIF-1 signaling pathway." To better understand the functions of the differentially expressed mRNAs described above, we performed GO and KEGG pathway enrichment analyses. Go annotations were obtained for approximately 499 differentially expressed mRNAs. GO analysis of these mRNA showed that they are involved in "immune system," "biological phase," and "cell killing" biological processes; are primarily enriched in "organelle part" and "macromolecular complex" cellular components; and are involved in molecular functions that include "translation regulator activity," "structural molecular activity," and "electron carrier activities" (Figure S3). In addition, KEGG annotation for 369 mRNAs were obtained ( Figure S4). The pathways with the most significant enrichment are shown in Figure 3. Remarkably, these mRNAs were enriched in "the ribosome pathway," "oxidative phosphorylation pathway," "Parkinson's disease pathway," and "Huntington's disease pathway" among others.

| Target gene prediction for lncRNA and qPCR validation
On the basis of the mode of action of lncRNAs and their target genes, we predicted the target genes for lnc-MKRN2-42:1 and GAS5:46. Compared with the healthy control group, BTD expression was approximately 71% lower and EIF4E expression was approximately 25% lower in the PD group, and the expression levels were positively correlated with lnc-MKRN2-42:1 ( Figure 4). Therefore, we selected lnc-MKRN2-42:1 and its target genes for further correlation analysis.

| D ISCUSS I ON
This study included 32 PD patients and 13 healthy controls. We quantitatively analyzed lncRNA expression in peripheral blood vesicles from PD patients and healthy controls using NGS and real-time quantitative PCR. We identified 15 upregulated and 24 downregulated lncRNAs, of which lnc-MKRN2-42:1 was selected for further study.
Bioinformatics analyses showed that lnc-MKRN2-42:1 could transregulate target genes such as BTD, EIF4E, MKNK1, and METTL5, involved in biological functions including apoptosis, synaptic remodeling, long-term potential, immunity, and glutamate neurotransmitter metabolism. Clinical correlation analyses showed that lnc-MKRN2-42:1 was positively correlated with MDS-UPDRS III scores for PD patients, suggesting that this lncRNA may be involved in the occurrence and development of PD. The process used to select lnc-MKRN2-42:1 is shown in Figure 6.
To ensure the experimental quality as far as possible, diseases that may cause changes in lncRNA expression levels were strictly excluded, and age and sex ratios were strictly defined in the selection of subjects for sequencing.   increase in miR-124 in cultured BV2 microglia and their associated exosomes. 26 Shi et al discovered that CNS-derived exosomes can efflux into blood and that the level of α-synuclein from CNS-derived exosomes in plasma is substantially higher in PD patients and is associated with PD severity. 27 Another study showed that exosomal miR-331-5p and miR-505 have diagnostic value for PD. 28 Recent studies have shown that lncRNAs are closely related to the pathogenesis of PD. High-throughput gene chip and sequencing screening results showed that a large number of lncRNAs are differentially expressed in brain tissues 13,29 and peripheral blood 11 of PD patients, PD animals, 30  The clinical manifestations of PD are complex. In addition to the typical movement disorder, balance impairments, restless leg syndrome, depression, anxiety, cognitive disorder, autonomic dysfunction, REM sleep behavior disorder, and speech disorder can occur.
Therefore, we used the MDS-UPDRS, H&Y stage, and Berg Balance Scale to evaluate dyskinesia, HAMA to detect anxiety, HAMD to detect depression, and MMSE and MoCA to detect cognitive impairment. 37 We analyzed the correlation between the results for most scales and lncRNA expression levels ( Figure S5). Other scales were only used to determine the severity of a patient's disease for more careful screening of the enrolled subjects. According to our results, The sample size in this study is insufficient, and validation in a larger sample is still needed. It must be noted that the plasma samples used in this study were only from clinically diagnosed PD patients and further studies will be necessary to evaluate lncRNA expression in the early stages of PD. Future research will need to investigate whether lnc-MKRN2-42:1 has biological functions in PD pathogenesis at the animal and cellular levels.
In conclusion, we discovered that peripheral blood exosomes contained PD-associated lnc-MKRN2-42:1, and its expression level was correlated with PD patients' clinical scores. Further research will explore whether lnc-MKRN2-42:1 is suitable as a new biomarker for PD.

CO N FLI C T S O F I NTE R E S T S
The authors have no conflicts of interest to disclose.