MiR‐193b deregulation is associated with Parkinson's disease

Abstract PGC‐1α/FNDC5/BDNF has found to be a critical pathway in neurodegeneration. MicroRNAs (miR(NA)s) are non‐coding regulatory RNAs whose dysregulation has been observed in multiple neurological disorders, and miRNA‐mediated gene deregulation plays a decisive role in PD. Here, candidate miRNA was chosen based on the literature survey and in silico studies. Chronic and acute models of PD were created using MPP+‐treated SH‐SY5Y cells. Twenty PD patients and 20 healthy volunteers were recruited. RT‐qPCR was performed to assess the expression of miRNA and genes. Severe mitochondrial dysfunction induced by acute MPP+ treatment instigated compensatory mechanisms through enhancing expression of PGC‐1α/FNDC5/BDNF pathway genes, while chronic MPP+ toxicity led to down‐regulated levels of the genes in SH‐SY5Y cells. PD peripheral blood mononuclear cells (PBMCs) also showed decreased expression of target genes. There were significant changes in the level of miR‐193b in both models, as well as PD PBMCs. Moreover, miR‐193b overexpression significantly affected PGC‐1α, FNDC5 and TFAM levels. Interestingly, down‐regulations of PGC‐1α, FNDC5, BDNF and TFAM were inversely correlated with miR‐193b up‐regulation in PD PBMCs. This study showed the deregulation of PGC‐1α/FNDC5/BDNF pathway in PD models and PBMCs, verifying its importance in neurodegeneration. Our findings also revealed that miR‐193b functions in PD development, possibly through regulating PGC‐1α/FNDC5/BDNF pathway, suggesting miR‐193b as a potential biomarker for PD diagnosis.


| INTRODUC TI ON
Parkinson's disease (PD) is a progressive neurodegenerative disorder (NDD) caused by the progressive and selective degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc) as well as accumulation of Lewy bodies containing α-synuclein in neocortex and brainstem. [1][2][3] While the precise mechanisms underlying pathogenesis of PD are not yet fully understood, several molecular mechanisms comprising oxidative stress, abnormal protein aggregation, mitochondrial dysfunction and neuroinflammation have been found to be strongly implicated in its pathogenesis, leading to programmed cell death of neurons. 3-7 1-methyl-4-phenylpyridinium (MPP+) is a complex I activity inhibitor extensively utilized to induce PD chemically due to its potential to create mitochondrial impairment followed by oxidative stress and apoptosis, all of which have close associations with PD pathogenesis. 2,4 Besides environmental factors playing an indispensable role in PD pathology, some susceptibility or causative genes and pathways have been discerned in PD, 1,7 Xi. 8 PGC-1α/FNDC5/BDNF pathway is one of them whose importance in neuroprotection as an effective target in neurological diseases and ageing has been highlighted. 4 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) has been identified as a master regulator of mitochondrial biogenesis and respiration that preserves neurons against degeneration. 4,7 It has been reported that PGC-1α overexpression positively regulates the expression of genes required for mitochondrial biogenesis such as TFAM and antioxidant capacity via rising expression of ROS-detoxifying enzymes, whereas PGC-1α silencing suppresses the expression of antioxidant enzymes as well as mitobiogenesis regulators. Thereby, PGC-1α pathway can be considered as a critical point of therapeutic intervention for PD and other neurological disorders. 4,9 Mitochondrial transcription factor A (TFAM) is a key modulator of the replication and transcription of mitochondrial DNA whose expression can be stimulated by PGC-1α. 10 Studies have shown possible connections between TFAM as a multifunctional regulator of mtDNA metabolism and PD pathogenesis. 11 There are many lines of evidence indicating that BDNF/TrkB is closely associated with pathophysiology of many NDDs [12][13][14] and enhanced expression of it has been demonstrated to defend striatal neurons against neurotoxicity in several PD models. 4 Brain-derived neurotrophic factor (BDNF) has been recognized as a versatile and multifunctional neurotrophin playing a vital role in a wide variety of neuronal functions ranging from neurogenesis and survival to synaptic plasticity by activating a particular receptor named tropomyosin receptor kinase B (TrkB). 12,15,16 TrkB is a membrane tyrosine kinase receptor mediating BDNF function which abounds in some brain regions where BDNF is highly expressed. 4,14 Numerous studies previously have shown that BDNF induces various intracellular signalling events via TrkB receptors, leading to an elevation in PGC-1α and TFAM expressions. 4,15 Fibronectin type III domain-containing protein 5 (FNDC5) is a PGC-1α-dependent myokine that induces the expression of neuroprotective agents including BDNF in the hippocampus, culminating in an elevated expression of PGC-1α in neurons. 4,16,17 Therefore, as our previous studies suggested, FNDC5 plays a beneficial role in neuronal survival, development, differentiation and resistance to neuronal stress and mitochondrial biogenesis. 4,17,18 MicroRNAs (miR(NA)s) are highly conserved endogenous small (~22 nucleotide) non-coding RNAs that control gene expression at the post-transcriptional level by binding to the 3' UTRs of mRNAs. 2,19,20 Multiple lines of evidence confirmed an essential role of miR-NAs in neuronal survival and normal function. 21,22 miRNAs participate extensively in a number of neurodegenerative processes like PD and their dysregulation has been observed in several PD models and post-mortem human brain tissues, resulting in their being introduced as attractive therapeutic targets or biomarkers for PD. 2,20,21,23,24 Therefore, our study is primarily aimed to assess miR-193b levels in PD patients' peripheral blood mononuclear cells (PBMCs) and SH-SY5Y cells injured acutely and chronically with MPP+, as in vitro models of the disease, in the hope of finding a potential candidate for PD biomarker. Furthermore, since altered expression of miRNAs can profoundly affect PD-related genes' expression and disturbed miRNA-mediated post-transcriptional regulation is deemed to be an important mechanism in neurodegeneration, 20,21 we checked the expression of PGC-1α/FNDC5/BDNF pathway components as miR-193b's potential targets in two PD models and PBMCs.  25 and verified by an expert neurologist. Twenty age-matched healthy volunteers without family history of PD or symptoms of NDDs were also recruited. In order to rule out any confounding factors before clinical assessment, the patients did not take anti-parkinsonian medications for at least 12 hours. Additional demographic and sample characteristics of PD and control patients are described in Table 1.

| Sample collection and separation of PBMCs
After getting the written informed consent, blood samples were drawn from the human donors in tubes containing EDTA (5 mL) and PBMCs were separated by the Lymphodex (Inno-Train) density gradient centrifugation. Fresh blood samples were diluted in PBS (1:1v/v), gently layered on the Ficoll solution at room temperature and centrifuged at 800 × g for 30 minutes. The PBMC-containing layer was collected and washed twice in PBS at 4°C (250 × g for 10 minutes) and the pellet (PBMCs) was stored at −70°C until further usage.  [28][29][30] were all served to predict a common miRNA targeting the PGC-1α/FNDC5/BDNF pathway components.

| Candidate miRNA selection and signalling pathway enrichment analysis
Literature mining was also carried out to find deregulated miRNAs in various NDDs. A human PD microarray data set (GSE16658) was analysed in this study (available on NCBI Gene Expression Omnibus (GEO)). The differential expression of array data has been done via limma 31 and GEOquery 32 packages in R. The clustering of differential expression of miRs was done and visualized by using the R package pheatmap. 33 Hierarchical clustering has been used in this study as the best method for analysing highthroughput expression data. Additionally, DianaTools MirPath v.3 34 was employed to visualize the signalling pathways in which miR-193b is implicated. miRTarBase 35 was used to find the secondary structure of pre-miR-193b. Using TargetScan 7.1 and miRmap, the putative miR-193b binding sites within five genes implicated in PGC-1α/FNDC5/BDNF pathway were retrieved.

| Cell transfection
MiR-193b mimic and miRNA negative control (scramble) were purchased from Dharmacon Inc SH-SY5Y cells were plated in 6-well plates and incubated until 80% confluence prior to transfection.

Transfection of oligonucleotides into SH-SY5Y cells was performed
with Lipofectamine 2000 reagent (Invitrogen) using 25 nM of miR-193b mimic and scramble, followed by treatment with 2000 µM MPP+ for 24 hours. Transfection efficiency was evaluated by qRT-PCR.

| MPP+ treatment and cell viability assessment
In brief, SH-SY5Y cells were seeded at a density of 2 × 10 4 cells/ cm 2 for acute and chronic MPP+ treatment. In acute MPP+ model, 24 hours after plating, the medium was replaced with the fresh medium with low serum containing 0-3000 µM MPP+ (Sigma-Aldrich) for 24 hours. To create chronic MPP+ model, the cells were exposed to a low-serum fresh medium containing the same concentrations of

| Detection of cell apoptosis by Annexin
The data are presented as mean ± SD. a P-value was calculated using Student's t test.
b P-value was calculated using the chi-square test.  to assess the diagnostic importance of the miR for discriminating PD patients from controls and its correlation with target genes. All data are presented as the mean ± standard deviation (SD) and collected from three independent experiments. P-value ≤.05 was considered to be statistically significant.

| Characteristics of study patients
Demographic characteristics and clinical data of all participants are presented in Table 1. Statistical analysis showed no significant disparities between two groups in terms of age and sex. prediction software led us to select miR-193b as an appropriate candidate for this study. Moreover, miR-193b showed a differential expression between PD patients' and healthy controls' PBMCs in microarray data set (GSE16658) ( Figure S1). To gain more insight into the importance of this miR in cellular processes, miRNA versus pathways heatmap was drawn. Interestingly, miR-193b was indicated to be highly relevant to the pathways associated with the nervous system such as neurotrophin TRK receptor signalling pathway and neurodegeneration-related pathways such as cell death, response to stress and intrinsic apoptotic signalling pathway ( Figure 2B). The secondary structure of pre-miR-193b and potential binding sites of the miR within 3′UTR of five genes' mRNAs implicated in PGC-1α/FNDC5/BDNF pathway are depicted in the illustration ( Figure S2).

| Characterization of acute and chronic MPP+ in vitro PD models
To evaluate the effect of acute toxicity on SH-SY5Y cells' survival, the cells were exposed to several concentrations of MPP+ PD models, rising from 15% and 21% to 36% and 52%, respectively ( Figure S3D).

| Expression of miR-193b and target genes in PD patients PBMCs
To investigate the expression of miR-193b and predicted mRNAs levels in PBMCs of 20 PD patients and 20 healthy controls, RT-qPCR analysis was performed. Similar to chronic PD model, there was a significant decrease in PGC-1α, TFAM, FNDC5, TrkB and BDNF expression in PD PBMCs as shown in Figure 5A, while PBMCs of patients with PD showed an increased level of miR-193b compared with controls ( Figure 5B).

| Diagnostic value of miR-193b
The usefulness of miR-193b in terms of differentiating PD patients from healthy controls, ROC curve and total area under the curve (AUC) were established. AUC value provides an estimate of the factor's potential to differentiate 2 groups by examining the sensitivity and specificity. The higher AUC value a marker has, the more effectively it can distinguish two conditions. The ROC curve analyses confirmed the significance of miR-193b level as a potential biomarker for PD diagnosis, with AUC value of 0.7925 (P = .0016, 95% CI: 0.6434-0.9416, Figure 6).

| D ISCUSS I ON
Although intensive research efforts have been made over the past decades to give insight into the pathomechanisms of PD, it still remains a major challenge in neurobiology. 6,19,36 Inaccessibility of patients' living target tissue and low availability or reliance on brain samples donated after death tend to impede significant advances in PD research, leading researchers to seek alternate windows into the biological basis of this disorder 20,37 , that is why numerous useful in vitro systems have been previously established to provide PD researchers with appropriate replica for simultaneously studying the mechanism relevant to PD and screening of pro-drugs. 2,20,38 Growing evidence also represents that there is a correspondence between peripheral blood and brain tissue, suggesting that evaluating disease-relevant genes' expression in peripheral blood can act as an advantageous proxy measure for gene expression in the CNS and reflect the state of illness in the brain. 37 PBMCs are deemed to be a peripheral laboratory providing a golden opportunity to study the mechanisms linked to NDDs and to find an indicator for their prognosis, diagnosis and treatment. 12,19 Accordingly, here, cell models of PD were developed to clarify whether miR-193b and PGC-1α/FNDC5/ BDNF is the most studied neurotrophin that modulates a wide repertoire of neuronal functions including neuronal morphology, survival, growth and differentiation as well as synaptic plasticity through interaction with its high-affinity receptor, TrkB. [12][13][14]45 BDNF/TrkB signalling axis is essential for the long-term survival of nigrostriatal system whose impairment is frequently observed in PD, contributing to the onset and progression of the disease. 12,14 In vitro and in vivo studies found that BDNF treatment protects DA neurons from multiple neurotoxin insults in various PD models. 4,12,20 Further, BDNF deficiency in CNS is known to have profound impacts such as synaptic dysfunction and impaired neuronal morphology, confirming the importance of this main trophic factor in various signalling pathways and circuits. 13 In agreement with our data showing upregulation of BDNF in response to acute MPP+, BDNF levels rose considerably in C57BL6 mice striatum 24 hours after administration of high dose of MPP+, according to Cunha et al. 46 By contrast, severe suppressed expression of BDNF along with its signalling axis regulator, TrkB, was clearly detected in acute MPTP-induced PD mice as well. 14 FNDC5 is a highly conserved transmembrane protein shown to be responsible for mediating the regulation of BDNF by PGC-1α. 4,16 In neuronal culture, elevating the levels of FNDC5 was found to reverse the apoptotic effects of Ab1−42 oligomers in Alzheimer's disease (AD) model and counteracts the suppression of BDNF expression by them. 16 However, knockdown of FNDC5 reduces BDNF expression in primary cortical neurons and impairs the development of neuronal precursors into mature neurons, representing an indispensable role of FNDC5 in neurons. 40 In the current study, acute MPP+ toxicity induced expression of FNDC5 in neuronal cells. To the best of our knowledge, this is the first study to examine the level of FNDC5 in acute model of PD. Collectively, the observed rise in target genes' levels can be a cellular adaptive response to MPP+ neurotoxicity to function as a compensatory protective strategy to attenuate destruction induced by neurotoxin. 7,19 Notably, only a limited number of PD studies were carried out using chronic models, leading us to also assess changes in gene expression in neuronal cells treated chronically with MPP+. We iden-  21 and AD. 51 miR-193b is a known mitomiR and redoximiR whose expression increases with age 55 , and has been introduced as a novel AD biomarker. 51 In the present study, we observed a sig- There were also lower levels of miR-193b levels in serum and plasma of patients with MCI and dementia of Alzheimer type (DAT). 51 In recent years, finding a promising biomarker for PD has been of utmost importance and blood miRNAs have always been one of the main considerations since they are not only easy handling and non-invasive, but also more useful than cytokines and growth factors found in serum or plasma. 53  To sum up, our findings suggest that MPP+ administration at low doses can create a more appropriate model of PD in comparison with acute MPP+ toxicity. Deregulation of PGC-1α/FNDC5/BDNF pathway was indicated in PD that may be a result of aberrant expression of miR-193b, verifying the importance of PGC-1α/FNDC5/BDNF pathway and miR-193b as well as their associations in PD pathology.
On top of that, this study provides evidence that miR-193b expression in PBMCs may be a potential diagnostic biomarker for PD and give insights into the disease diagnosis. However, further studies must be conducted to confirm diagnostic value of miR-193b and to fully comprehend mechanisms by which this miR operates on target genes in PD.

ACK N OWLED G EM ENTS
We would like to show our gratitude to our colleagues especially Ghaedi as the P. I.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.