AVE0991 regulates HOTAIRM1/miR-223-3p/α-synuclein to improve behaviour and protect dopaminergic neurons in hu-man α- syn (A53T) overexpressing mice

AVE0991 regulates HOTAIRM1/miR-223-3p/α-synuclein to improve behaviour and protect dopaminergic neurons in hu-man α- syn (A53T) overexpressing mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article AVE0991 regulates HOTAIRM1/miR-223-3p/α-synuclein to improve behaviour and protect dopaminergic neurons in hu-man α- syn (A53T) overexpressing mice Rui Duan, Liang Shi, Yang Deng, Jiang Wu, Shiyao Wang, Qiang Peng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4723150/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Parkinson's disease (PD) is a prevalent type of neurodegenerative disorders. AVE0991, a non-peptide analogue of Ang-( 1 – 7 ), by which the progression of PD has been discovered to be ameliorated, but the specific mechanism whereby AVE0991 modulates the progression of PD re-mains unclear. The mice overexpressing of human α-syn (A53T) were established to simulate PD pathology, and we also constructed an in vitro model of mouse dopaminergic neurons overexpressing hα-syn (A53T). The [ 18 F] FDG-PET/CT method was also employed to assess FDG uptake in human α-syn (A53T) overexpressing mice. Levels of lnc HOTAIRM1, miR-223-3p were detected via qRT-PCR. Flow cytometry was deployed to assay cell apoptosis. Here, we found that AVE0991 improved behaviour disorder and decreased α-syn expression in the substantia nigra in mice with Parkinson's disease. AVE0991 inhibited apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) by lncRNA HOTAIRM1. MiR-223-3p binds to HOTAIRM1 as a ceRNA and directly targets α-syn. Our present study shows that the angiotensin-( 1 – 7 ) analogue AVE0991 targeted at the HOTAIRM1/miR-223-3p axis to degrade α-synuclein in PD mice, and showed neuroprotection in vitro. Biological sciences/Cell biology Biological sciences/Molecular biology Biological sciences/Neuroscience Health sciences/Diseases Health sciences/Neurology Parkinson's disease AVE0991 α-syn HOTAIRM1 miR-223-3p Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Parkinson's disease (PD) is the 2nd prevalent neurodegenerative disease and is expected to impose heavy stresses and economic burdens on society as the population ages 1 . Treatment of PD includes pharmacologic approaches associated with levodopa preparations, nonpharmacologic approaches (such as exercise and physical, occupational, and speech therapies) and deep brain stimulation 2, 3 . However, all above methods only provide symptomatic relief without delaying the progression of the disease 4 . The renin-angiotensin system (RAS), which is widely distributed in various brain regions, includes the ACE2/Ang-( 1 – 7 )/ Mas axis 5 . As found in prior studies, Ang-( 1 – 7 ) is found in the hippocampus, thalamus and substantia nigra, and has beneficial roles in multiple neurological disorders, including PD 6, 7 . For example, Ang-( 1 – 7 ) attenuates blood-brain barrier damage following cerebral ischemia-reperfusion by regulating TIMP-1/MMP-9 8 . Ang-( 1 – 7 ) decreases inflammation levels and significantly improves cognitive outcomes in traumatic brain injury (TBI) mice 9 . Meanwhile, in 6-hydroxydopamine Parkinsonian rats, Ang-( 1 – 7 ) could inhibit HMGB-1/RAGE/NF-κB signaling to ameliorate dyskinesia 10 . Notably, Ang-( 1 – 7 ) exhibits an extremely short half-life (10–20 s) and must be administered intensively to act in the nervous system 11 . Therefore, we chose AVE0991, the non-peptide analogue of Ang-( 1 – 7 ), for intraperitoneal administration to conduct our study. Our previous researches have demonstrated that, AVE0991 could reduce neuroinflammation and exerts protective effects in both AD mouse model and mouse model of accelerated aging 12, 13 . However, no study has clarified whether AVE0991 regulates the progression of PD and the specific mechanism. Based on the genetic characteristics, Parkinson’s disease can be divided into familial and sporadic types 14 . Numerous researches have indicated that α-synuclein (α-syn) acts as an important pathogenetic agent in both forms of the disease over the past decades. Intraneuronal proteinaceous cytoplasmic inclusions, which are pathologically characteristic of PD, cause α-syn deposition 15 . Additionally, the A53T mutation of NG_011851 (SNCA) gene encoding hα-syn can lead to autosomal-dominant PD 16 . Our preliminary research shows that Ang-( 1 – 7 ) decreases α-syn deposition in Parkinson's disease 17 , yet the underlying mechanism is still unclear. MiRNAs, as a sort of non-coding RNA, which have been recognized to be involved in the developmental process of several neurological diseases 18 . For instance, miRNA-326 was found to diminish tau phosphorylation and neuron apoptosis in Alzheimer's disease by targeting VAV1 19 . Moreover, miR-96-5p alleviates cerebral ischemia-reperfusion injury in mice by inhibiting pyroptosis via downregulating caspase-1 20 . In addition, our previous research has found that AVE0991 alleviates neuroinflammation in AD-like mouse brain by regulating miR-223-3p related pathways, above of which also demonstrates that AVE0991 has a strong neuroprotective property. Meanwhile, the levels of plasma miR-223-3p were significantly decreased in PD patients 21, 22 . Accordingly, we inquired into whether AVE0991 could be functional in PD through the regulation of miR-223-3p and its specific mechanism. Similarly, another non-coding RNA, lncRNAs, have been also stated that participated in a variety of neurological diseases 23–25 . Theoretically, lncRNAs could sponge miRNAs as competitive endogenous RNAs (ceRNAs) and thus regulate target protein levels 26 . For example, lncRNA-AK046375 suppressed oxidative stress by up-regulating MT2 expression through the sequestration of miR-491-5p and thus attenuated neurological impairment after TBI 27 . LncRNA BIRF could promote cerebral ischemia tolerance induced by cerebral ischemia preconditioning through sponge miR-330-5p up-regulation of GLT-1 28 . Additionally, lncHOTAIRM1 (HOXA transcript antisense RNA myeloid-specific 1) was observed to be significantly elevated in circulating leukocytes in PD patients, and overexpression of HOTAIRM1 increases 6-hydroxydopamine-induced apoptosis in SH-SY5Y cells 29 . Nonetheless, it is still unknown about the explicit mechanism of HOTAIRM1 in PD. In this work, we addressed whether AVE0991 exerts neuroprotective effects through HOTAIRM1/miR-223-3p to modulate α-syn in PD models in vitro and in vivo. The study also provided novel diagnostic indicators and drug targets for treatment of PD. Materials and methods Ethical statement All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the ethical review Committee of Nanjing First Hospital (Number: KY20190509-05), and written informed consent was obtained from all subjects. Animals and Treatment Male C57BL/6J mice (weight 20-25 g) were received from Vital River Laboratories (Beijing, China). All mice were kept in chambers at controlled ambient temperature and relative humidity with permission to drink and eat freely. The study was permitted by the Animal Care and Use Committee of Nanjing First Hospital (Number: DWSY-22003128). All mice studies were performed in accordance with the ARRIVE guidelines for reporting animal experiments. Mice were randomized into three experimental groups: Control group, hα-syn (A53T) group, hα-syn (A53T) + 10 mg/kg AVE0991 group. All mice were intraperitoneally injected with sterile saline (0.9%) or AVE0991 (10 mg/kg, MedChemExpress LLC, NJ, USA) once daily for 30 continuous days. Stereotaxic Injection of AAV- HΑ-Syn (A53T) Vector The mice (8 weeks old) were anesthetized with 2% isoflurane in O 2 (RWD Life Science, Shenzhen, China) and then stabilized on a stereotaxic instrument (David Kopf Instruments). AAV-hα-syn (A53T) (120 nl - 7.0 ×10 12 vg/ml) was injected at 0.4 μl/min into the right SNpc of mice (AP = −2.9 mm, L = −1.3 mm, DV = −4.5 mm). Notably, the pipette was kept for 5 min post-injection before being slowly withdrawn. LC-MS/MS LC-MS/MS was performed to determine the amount of AVE0991 in brain of mice injected with AVE0991. Brain tissue was homogenized with 500 μL saline, centrifuged and then transferred 100 μL homogenized supernatant to a tube and reconstituted with 500 μL acetonitrile (Sigma-Aldrich, USA). After centrifugation again, 5 μL of supernatant was taken for sampling. MS spectra were collected from m/z 581.72→308.1. MS/MS profile was obtained with 43 V collision energy. Data were visualized and analyzed using MultiQuant-MD 3.0.3 (AB Sciex, FC, USA). Rotarod Test The rotarod test was utilized to evaluate the balance and motor coordination of mice. Mice were kept on a rotating device (IITC Life Science, CA, USA) with the speed adjusted from 5 rpm to 40 rpm for 300 s. Recording of the time each mouse dropped from the rotarod was performed. Testing was performed 3 times per mouse, at least 30 minutes apart. Open Field Test Open field test was performed to assess the general locomotor activity of mice. Mice were located in the center of an open field square (47 cm× 47 cm × 40 cm) for 5 min. During this period, a TSE software (TSE Systems, Bad Homburg, Germany) was used to analyze the exercise time of mice. PET/CT Imaging Mice were scanned for [ 18 F] FDG PET 30 days after AVE 0991 injection. Briefly, mice were fasted for 6 h and then injected with [ 18 F] FDG through the tail vein. 45 minutes later mice were anesthetized with 2.5% isoflurane, fixed, and then subjected to PET scanning acquisition (10 minutes, Inveon, Siemens) as well as CT scanning. Maximum standardized uptake values were computed to estimate [ 18 F] FDG uptake content. Immunohistochemistry SN-containing mouse brain sections were deparaffinized, hydrated, antigenically repaired, and permeabilized using 0.5% Triton X-100, subsequently blocked with 5% bovine serum albumin at room temperature, and then incubated with primary antibody α-syn (1:100, ab212184, Abcam, MA, USA) at 4 °C overnight. On the next day, sections were incubated with biotinylated secondary antibody IgG (1:200, ab64256, Abcam, MA, USA) at room temperature. Sections were stained with diaminobenzidine and restained with hematoxylin, dehydrated and sealed, and images were obtained by a fluorescence microscope (Olympus Corporation, Tokyo, Japan). LncRNA Sequencing and Bioinformatic Analysis Total RNA was extracted from the right SNpc tissues of mice using TRIzol reagent (Thermo Fisher Scientific) and its concentration and quality were measured by Qubit fluorometer (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. The quality of data processing of raw reads was checked by FastQC-v0.11.8, and low-quality bases and adaptors contamination were trimmed with Fastp-v0.19.5 if necessary. RNA library preparation was performed according to the manufacturer’s instructions, using 1 μg of DNase-treated total RNA as input. The libraries were quantified by using an ABI 9700HT Fast Real-Time PCR system (Thermo Fisher Scientific). Samples were sequenced using the NovaSeq platform (Illumina) to generate 150 bp paired-end reads. STAR-v2.7.7a was performed to map clean reads to the reference genome. Normalization of mapped reads and differential analysis of lncRNAs were performed using DESeq2-v1.10.1. Parameters |log2FC| ≥1 and P < 0.05 was defined significantly differentially expressed lncRNAs. Sequencing database has been archived in GenBank (GSE244050). qRT-PCR Total RNA was obtained from tissues or cells using Trizol reagent (Thermo Fisher Scientific). RNA was then reversely transcribed to cDNA with a kit (ABM, Richmond, BC, Canada) following measuring the concentration. Next, it was detected using SYBR-Green method on an ABI7500 type sequence detection system (7500, ABI, USA). U6 and β-actin were used as internal references for miRNA and mRNA, respectively. The specific primer sequences for qRT-PCR were shown in Table S1 . Primary Dopaminergic Neurons Culture and Transfection Fetal mice were dissected from mice at 15 days of gestation and the midbrain tissue was isolated. The tissue was then digested with 0.25% trypsin for 10 minutes at 37°C and the cells were precipitated by filtration and centrifugation. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded in plates pre-coated with poly-L-lysine. After incubation for 4 hours at 37 °C in a 5% CO 2 incubator, the medium was replaced with Neurobasal medium containing 2% B27 and incubation was continued for about 8 days with fluid changes every 2 days. The final concentration of AVE0991 used in the experiments was 1×10 -6 M. Briefly, miR-223-3p mimic, miR-223-3p inhibitor, and respective negative controls (NC mimic, NC inhibitor) were acquired from Shanghai GenePharma Company, hα-synuclein (A53T), lenti-HOTAIRM1, sh-HOTAIRM1 and their lentiviral scramble control shRNA were adopted from Shanghai GeneChem Company. Based on the manufacturer’s protocol, transfected them to cells with Lipofectamine 3000. Lentivirus infection was performed by following the instructions, while puromycin was then added to screen for stable cells. The expression of target genes or protein was verified by qPCR or Western blot. Western Blot Total proteins were extracted from mouse midbrain tissues or cells using RIPA lysate (Beyotime, Shanghai, China) and protein concentration was quantified by a BCA kit (Thermo Scientific, San Jose, CA, USA). After mixing and heating to denaturation by adding an appropriate amount of loading buffer, the samples were separated by sodium dodecyl sulfate-polyacrylamide gels electrophoresis. Following transfer to the membrane it was blocked in 5% non-fat milk at room temperature. This was followed by overnight incubation at 4 °C with the following primary antibodies: anti-α-syn (1:1000, ab212184, Abcam, MA, USA), anti-bcl2 (1:1000; ab196495; Abcam, MA, USA), anti-bax (1:1000; #2772; CST, MA, USA), anti-caspase-3(1:1000; #9661; CST, MA, USA) and β-actin (1:1000; #4970; CST, MA, USA). The membranes were washed 3 times and then incubated with corresponding HRP labeled IgG at room temperature. Lastly, strip visualization using an ECL kit (Thermo Fisher Scientific) and quantified by analysis using Image J (National Institutes of Health, Bethesda, Maryland, USA) Annexin V/Propidium Iodide (PI) Apoptosis Staining The apoptosis rate of the cells was detected by flow cytometry. Cells were collected, centrifuged, washed, and resuspended with 500 μL buffer. In compliance with the instructions of the apoptosis detection kit (KGA107, Keygen,China) the cells were stained for 15 min, and the proportion of apoptotic cells was quantitatively analyzed by flow cytometry (FACSCalibur, BD Biosciences, CA, USA). Fluorescent in Situ Hybridization (FISH) Frozen sections of brains were subjected to in situ hybridization using the HOTAIRM1 and miR-223-3p probe (RiboBio Co., Ltd, Guangzhou, China) according to the protocol described. After hybridization, the sections were blocked with blocking buffer. Then, the sections were incubated with anti-TH (1:100; #45648, Cell Signaling Technology) overnight at 4 °C, and after washing 3 times in PBS, the sections were incubated with Cy5-labeled goat anti-mouse IgG (1:200; GB27301, Servicebio, Wuhan, China) at room temperature. 4ʹ,6-Diamidino-2-phenylindole staining was completed and the sections were blocked and visualized using a microscopy. Dual Luciferase Reported Assay The potential sites of α-syn 3’UTR binding to miR-223-3p and assessment of miR-223-3p binding to Lnc HOTAIRM1 were analyzed by online bioinformatics database TargetScan and bioinformatics software RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/). The sequences of luciferase reporter plasmids HOTAIRM1-WT and HOTAIRM1-Mut were constructed and cells were co-transfected with plasmids and NC mimic or miR-223-3p mimic with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were collected after 48 h, lysed and assayed for luciferase activity by the dual luciferase reporting and detection system (Promega, WI, USA). The same procedure was used to determine whether α-syn and miR-223-3p were bound. Patients and Samples Blood samples were collected from PD patients and healthy controls admitted to Nanjing First Hospital from 2021 to 2022. No significant differences were found in the age and gender of the participants. The study was approved by the ethical review Committee of Nanjing First Hospital (Number: KY20190509-05). All participants signed a written informed consent before participating in this study. The upper plasma layer was separated by centrifugation (2,500 g, 15 min) within a short period of time after blood samples were collected and the level of α-syn in plasma was measured by enzyme-linked immunosorbent assay (ELISA) kit (No. JL12231, Jianglai Biologicals, Shanghai, China). The lower samples were extracted for RNA according with the protocol of the manufacturer's instructions, and the expression of miR-223-3p was determined by qRT-PCR as described above. Clinical parameters are detailed in Table S2. Statistical Analysis All statistical analyses were performed with the Prism 8.0 (GraphPadSoftware, Inc., La Jolla, USA) software. Differences were analyzed by Student's t-test for both groups or one-way ANOVA between multiple groups. Correlations analysis of blood samples data was evaluated by the Pearson’s method. Data are presented as mean ± SD of at least three independent experiments. Statistical significance was considered if the P < 0.05. Results AVE0991 improves behavior disorder and decreased α-syn expression in the hα-syn (A53T) mice AVE0991 was measured in the brain of mice after intraperitoneal injection using LC-MS/MS. The characteristic peak of AVE0991 was detected by full-scan MS signals in the brains of administered mice (Fig. 1A) . This suggests that AVE0991 can be penetrate into the brain by intraperitoneal injection. During the rotarod test, mice infected with hα-syn (A53T) virus spent notably less time on the stick than control mice. However, AVE0991 increased stick retention time in mice infected with hα-syn (A53T) virus after administration (Fig. 1B) . Mice were tested for spontaneous locomotion via the open-field test, which showed that mice infected with hα-syn (A53T) virus had remarkably reduced spontaneous motility compared with control mice. However, AVE0991 increased spontaneous activity in A53T mice after administration of AVE0991 (Fig. 1C) . Subsequently, whole-body PET/CT scans revealed that, as expected, [ 18 F] FDG excretion occurred primarily in the urinary bladder, while the midbrain manifested high levels of [ 18 F] FDG uptake, and compared to control mice, [ 18 F] FDG uptake in the midbrain region of mice infected with hα-syn (A53T) virus was conspicuously reduced, while [ 18 F] FDG uptake in the midbrain region of mice infected with hα-syn (A53T) virus was increased after administration of AVE0991 (Fig. 1D) . Besides, immunohistochemical analysis indicated that α-syn expression was elevated in the substantia nigra of mice infected with hα-syn (A53T) virus compared with the control group, whilst AVE0991 administration relatively reduced α-syn deposition in the substantia nigra of mice infected with hα-syn (A53T) virus (Fig. 1E) . These results suggested that AVE0991 ameliorated behavioral deficits and decelerated α-syn accumulation in the substantia nigra of hα-syn (A53T) mice. LncRNA HOTAIRM1 induces apoptosis in hα-syn (A53T)-overexpressing dopaminergic neurons LncRNAs have been described to be strongly associated with a range of neurological disorders. To investigate whether lncRNAs are involved in AVE0991 therapeutic effects on mice infected with hα-syn (A53T) virus, we carried out high-throughput lncRNA sequencing of right SNpc tissues in mice infected with hα-syn (A53T) virus with or without AVE0991 treatment. As illustrated in Fig. 2A , 19 differentially expressed lncRNAs were identified in total after AVE0991 treatment, with corrected thresholds of P 1.5. Of those, 12 lncRNAs were prominently up-regulated, whereas the other 7 lncRNAs were notably down-regulated. Our preliminary study shows that elevated levels of HOTAIRM1 expression in the blood of PD patients was elevated 30 . Meanwhile, the HOTAIRM1 levels in right SNpc of mice overexpressing hα-syn (A53T) after administration of AVE0991 were further confirmed by qRT-PCR (Fig. 2B) . Then, we further knocked down or overexpressed HOTAIRM1 in primary dopaminergic neurons with hα-syn (A53T) overexpression (Fig. 2C) . Western blot findings indicated that α-syn expression was clearly increased in neurons that overexpressed HOTAIRM1, while α-syn expression was decreased by knocking down HOTAIRM1 compared with the corresponding control group (Fig. 2D , E) . We determined neuronal apoptosis using flow cytometry. The data suggested that HOTAIRM1 overexpression increased apoptosis in primary dopaminergic neurons overexpressing hα-syn (A53T) compared with the corresponding controls, while knockdown of HOTAIRM1 relatively reduced the apoptosis rate (Fig. 2F , G) . Meanwhile, we further examined several apoptosis-related proteins. Western blot analysis disclosed that compared with the corresponding controls, the expression of Bcl2 was obviously down-regulated and the expression of Bax and cleaved caspase-3 was markedly up-regulated in primary dopaminergic neurons overexpressing HOTAIRM1, while knockdown of HOTAIRM1 upregulated Bcl2 expression and decreased Bax and cleaved caspase-3 expression (Fig. 2H-K) . All above-mentioned findings suggested that lncRNA HOTAIRM1 induced apoptosis in hα-syn (A53T)-overexpressing dopaminergic neurons. AVE0991 inhibits the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) by a lncRNA HOTAIRM1-manner Subsequently, to further investigate whether AVE0991 affects the pathological progression of α-syn via HOTAIRM1, we examined the relative expression of α-syn in hα-syn (A53T)-overexpressing primary dopaminergic neurons. Western blot analysis demonstrated that AVE0991 treatment down-regulated α-syn expression in primary dopaminergic neurons infected with hα-syn (A53T) virus compared with the corresponding controls, whereas overexpression of HOTAIRM1 reversed the reduction of α-syn induced by AVE0991 treatment (Fig. 3A, B). Meanwhile, we probed the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) by flow cytometry. The results showed that AVE0991 treatment reduced apoptosis in primary dopaminergic neurons compared with controls, while overexpression of HOTAIRM1 reversed the protective effect of AVE0991-induced neurons (Fig. 3C, D). Furthermore, Western blot data illustrated that, compared with controls, AVE0991 treatment in primary dopaminergic neurons overexpressing hα-syn (A53T) showed that Bcl-2 expression was upregulated and Bax expression was downregulated, while cleaved caspase-3 expression was downregulated, while further overexpression of HOTAIRM1 reversed the above results (Fig. 3E-H). The mentioned results demonstrated that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) through lncRNA HOTAIRM1. miR-223-3p binds to HOTAIRM1 as ceRNA and directly targets α-syn to inhibit apoptosis in dopaminergic neurons overexpressing hα-syn (A53T) Firstly, we used bioinformatics software RNAhybrid and TargetsScan to identify potential miRNAs that could bind to HOTAIRM1. The analysis showed that the sequence of miR-223-3p is capable of complementary binding to the 3′-UTR sequence of HOTAIRM1 and α-syn (Fig. 4A-C) . Next, we performed subcellular localization of several related molecules by FISH assay. The results indicated caspase-3 that in mouse dopaminergic neurons infected with hα-syn (A53T) virus, HOTAIRM1 was mainly in the neuronal cytoplasm and miR-223-3p was also co-localized with HOTAIRM1 in the cytoplasm (Fig. 4D) . Thus, we hypothesized that miR-223-3p might bind to HOTAIRM1 as ceRNA, thereby further regulating the expression of target genes after transcription. To validate further the relevance between miR-223-3p and HOTAIRM1, HOTAIRM1 was overexpressed or knocked down in primary dopaminergic neurons infected with hα-syn (A53T) virus and then detected miR-223-3p levels with qRT-PCR. The outcomes displayed that overexpression of HOTAIRM1 suppressed miR-223-3p levels, while knockdown of HOTAIRM1 increased miR-223-3p levels compared with the corresponding controls (Fig. 4E) . Subsequently, the test results for the dual luciferase reporter gene assay were met with expectations. miR-223-3p dramatically decreased HOTAIRM1-WT luciferase activity with no reduction in HOTAIRM1-mut activity (Fig. 4F) . Also, the dual luciferase reporter gene assay exhibited that miR-223-3p greatly decreased α-syn-WT luciferase activity, yet not α-syn-MUT activity (Fig. 4G), which also indicated that α-syn is a target protein of miR-223-3p. Next, we transfected miR-223-3p mimics or miR-223-3p inhibitors in dopaminergic neurons of mice overexpressing hα-syn (A53T) (Fig. 4H) . Western blot results identified that α-syn expression was declined in neurons overexpressing miR-223-3p compared to the corresponding controls, while miR-223-3p knockdown relatively increased α-syn expression (Fig. 4I, J) . We examined the apoptosis of neurons by flow cytometry. It was found that miR-223-3p mimics transfection reduced apoptosis induced by infection with hα-syn (A53T) virus compared with the corresponding controls, while miR-223-3p inhibitors transfection relatively exacerbated the rate of apoptosis (Fig. 4K, L) . Meanwhile, we further examined several apoptosis-related proteins. Western blot analysis showed that, compared with the corresponding control group, the expression of Bcl2 was up-regulated in neurons overexpressing miR-223-3p, the expression of Bax was down-regulated, and cleaved caspase-3 expression was down-regulated, whereas knockdown of miR-223-3p was reversed (Fig. 4M-P) . To summarize, these results prove that miR-223-3p acts as a ceRNA binding to HOTAIRM1 and directly targets α-syn to inhibit the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T). AVE0991 inhibits the apoptosis of dopaminergic neurons overexpressing A53T α-syn by up-regulating miR-223-3p We further explored whether AVE0991 inhibited α-syn expression through miR-223-3p. qRT-PCR results displayed that compared with the control group, treatment of AVE0991 increased the levels of miR-223-3p in right SNpc of mice overexpressing hα-syn (A53T) (Fig. 5A) . Western blot demonstrated that AVE0991 treatment decreased α-syn expression in primary dopaminergic neurons of overexpressed hα-syn (A53T) mice compared with the corresponding control group, while transfection of miR-223-3p inhibitors reversed the decrease of α-syn elicited by AVE0991 treatment (Fig. 5B, C) . Meanwhile, the apoptosis of primary dopaminergic neurons overexpressing hα-syn (A53T) was detected by flow cytometry. Results showed that treatment of AVE0991 reduced neuronal apoptosis, while transfection of miR-223-3p inhibitors reversed this effect of AVE0991 (Fig. 5D, E) . Western blot analysis indicated that, compared with the control group, following administration of AVE0991, primary dopaminergic neurons overexpressing hα-syn (A53T) had noticeably up-regulated expression of Bcl2, down-regulated expression of Bax and cleaved caspase-3. Further miR-223-3p inhibitors transfection reversed the above results (Fig. 5F-I) . These findings suggested that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) through upregulation of miR-223-3p expression. Relative plasma levels of miR-223-3p in PD patients are significantly correlated with levels of α-syn Finally, with the purpose of further clarify the clinical significance of miR-223-3p and α-syn as biomarkers of PD, plasma was collected from PD patients and healthy individuals and then measured the levels of miR-223-3p and α-syn. Analysis of qRT-PCR resulted in decreased expression of miR-223-3p in the plasma of PD patients compared to healthy controls (Fig. 6A) . Meanwhile, compared to healthy controls, plasma α-syn expression in PD patients was relatively elevated (Fig. 6B) . The above results were similar to the trend of miR-223-3p and α-syn expression levels in animal and cellular models. Then, we evaluated the correlation between miR-223-3p and α-syn levels in plasma of PD patients, finding that there was a significant negative correlation between the plasma levels of miR-223-3p and α-syn expression in PD patients (Fig. 6C) . Discussion PD, one of the most common synucleinopathies, are pathologically characterized by aggregated α-synuclein riboproteins forming neurons or glial intracytoplasmic inclusions 31, 32 . It is identified that the SNCA gene was discovered to contain a single point mutation leading to a non-synonymous amino acid substitution in p.A53T, resulting in familial early-onset PD 33, 34 . These also suggest that lowering the expression of α-syn may be an effective therapeutic option for Parkinson's disease-like disorders. Therefore, we used a mouse model in which PD-like pathology was evoked by overexpression of human α-syn (A53T) in dopaminergic neurons in this study, which can help understand progression of PD. Our results demonstrate that mice infected with hα-syn (A53T) virus exhibited the basic characteristics of PD, including significantly reduced time spent rotating rods and decreased spontaneous activity, compared with control mice. Moreover, α-syn accumulation increased in the substantia nigra of mice infected with hα-syn (A53T) virus. In addition to α-syn deposition, loss of dopaminergic neurons in the nigra, especially in the ventral-lateral portion, is also a pathological signature of PD 35 . Therefore, we also constructed an in vitro model of mouse dopaminergic neurons overexpressing hα-syn (A53T) for further study. Since decades ago, the Ang-(1-7) non-peptide analogue AVE0991 was already found to play an influential task in diseases of various systems. For example, it has been reported that AVE0991 has a cardioprotective effect under hyperglycemia conditions 36 . Murphy et al. found that AVE0991 has certain antitumorigenic and anticachectic actions, suggesting it may be useful in the treatment of cancer or other severe muscular dystrophy conditions 37 . Meanwhile, AVE0991 was proven that can ameliorate arthritic damage by diminishing neutrophil infiltration and cytokine generation 38 . In the study, our findings demonstrated that AVE0991 can alleviate the behavioural disorder of hα-syn (A53T) overexpressing mice, and at the same time, AVE0991 can relatively reduce the apoptosis of mice dopaminergic neurons overexpressed hα-syn (A53T). Emerging evidence suggested that miRNAs contribute to the pathogenesis of PD 26 . A host of miRNAs are differentially expressed in PD and impact the process, including miR-153-3p 39 , miR-155 40 , and miR-101 41 , etc. Of special note, it was found that miR-7a-5p can target and modulate the α-syn expression, the marker of PD, in mouse dopaminergic neurons 42 . Su et al. testified that inhibition of miR-26a expression leads to dopaminergic neuronal cell death, and motor deficits in normal mice 43 . It is worth emphasizing that in the present research, we verified miR-223-3p directly targets α-syn for inhibiting apoptosis in dopaminergic neurons overexpressing hα-syn (A53T). AVE0991 restrained apoptosis in dopaminergic neurons overexpressing A53T α-syn by up-regulating miR-223-3p. A growing body of research has suggested that lncRNAs perform an essential job for neurodegenerative conditions, including PD 44 . Commonly, in neurological diseases, lncRNA can affect the target RNA levels, and a break in the balance between lncRNA and target RNAs can cause changes in neuronal viability and induces the survival or death of neurons. For example, it is reported that elevated expression of lncRNA BDNF-AS promotes MPTP-induced apoptosis and autophagy in PD model mice 45 . It has been observed that repression of lncRNA UCA1 expression reduces MPP + -induced inflammation and oxidative stress in PD cells, thereby alleviating dopaminergic neuronal damage 46 . In light of the above findings, we then explored the participation of lncRNAs in regulation of the progression of PD after administration of AVE0991 in mice with hα-syn (A53T) overexpression. Sequencing outcomes combined with qRT-PCR data showed that in dopaminergic neurons of AVE0991-treated hα-syn (A53T) overexpressing mice, the lncRNA HOTAIRM1 was down-regulated. In vitro, knocking down HOTAIRM1 decreased α-syn expression while overexpression of HOTAIRM1 reversed the α-syn reduction induced by AVE0991 administration. This observation indicated that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) via a HOTAIRM1-dependent manner. More and more reports have indicated that the subcellular localization of lncRNA may also affect their biological functions 47, 48 . Numerous lncRNA are distributed in cytoplasm and nucleus, but their distribution is different 49 . In general, lncRNA widely distributed in the cytoplasm mainly affect cell signalling cascades and regulate the stability or translation process of their target mRNA, while lncRNA in the nucleus are involved in RNA processing, transcriptional regulation, and chromatin interactions 50, 51 . Here, FISH testing revealed that HOTAIRM1 was mainly located in the cytoplasm of mouse dopaminergic neurons and co-localized with miR-223-3p in the cytoplasm, suggesting that HOTAIRM1 may have the function of competitively binding to miRNA. Bioinformatics prediction and luciferase reporter gene detection showed that HOTAIRM1 interacts with miR-223-3p. In addition, HOTAIRM1 down-regulates the miR-223-3p expression. Based on the above data, we speculated that miR-223-3p can competitively bind HOTAIRM1 and directly act on the downstream target α-syn to exert vital effects in the cytoplasm of dopaminergic neurons. Our present study shows that the angiotensin-(1–7) analogue AVE0991 targeted the HOTAIRM1/miR-223-3p axis to degrade α-syn in mouse model with PD-like pathology is induced by overexpression of human α-syn (A53T), and showed neuroprotection in vitro. Here, the study highlights the valuable neuropharmacological benefits of AVE0991 in the course of PD disease. Furthermore, this study supports essential evidence for PD treatment strategies targeting α-syn. These findings expand our understanding that the angiotensin-(1–7) analogue AVE0991 has a strong potential for PD therapy and provides support for the discovery of therapeutic approaches for PD and other related synucleinopathies. Declarations Author contributions Rui Duan designed and performed most experiments, analyzed data, and prepared the manuscript; Liang Shi and Yang Deng performed experiments and analyzed the data. Jiang Wu and Shiyao Wang helped with the mice model construction. Qiang Peng, Zhongyuan Li and Zhaohan Xu performed experiments; Feng Wang, Xue Xue, and Qing Gao directed the study and prepared the manuscript. All authors read and approved the final manuscript. Conflicts of interest The authors declare no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2022YFC2406900), the National Natural Science Foundation of China (82301609), China Postdoctoral Science Foundation (2022M711666), China Postdoctoral Science Foundation (2023M741764), Natural Science Foundation of Jiangsu Province (BK20220196), the Medical Science and Technology Program of Nanjing (JQX22006), the International Joint Research and Development Project of Nanjing (202201030), the International Joint Research and Development Project of Nanjing (202308005) and Xinghuo Talent Program of Nanjing First Hospital. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References de Lau, L.M. & Breteler, M.M. Epidemiology of Parkinson's disease. Lancet Neurol . 5 (6), 525-535 (2006). Mao, Q., Qin, W.Z., Zhang, A. & Ye, N. 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Tau accelerates α-synuclein aggregation and spreading in Parkinson's disease. Brain . 145 (10), 3454-3471 (2022). La Vitola, P. et al. Peripheral inflammation exacerbates α-synuclein toxicity and neuropathology in Parkinson's models. Neuropathol Appl Neurobiol . 47 (1), 43-60 (2021). Smajić, S. et al. Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state. Brain . 145 (3), 964-978 (2022). Ebermann, L. et al. The angiotensin-(1-7) receptor agonist AVE0991 is cardioprotective in diabetic rats. Eur J Pharmacol . 590 (1-3), 276-280 (2008). Murphy, K.T. et al. Mas Receptor Activation Slows Tumor Growth and Attenuates Muscle Wasting in Cancer. Cancer Res . 79 (4), 706-719 (2019). da Silveira, K.D. et al. Anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, MAS, in experimental models of arthritis. J Immunol . 185 (9), 5569-5576 (2010). Zhang, J. et al. LncRNA miR-17-92a-1 cluster host gene (MIR17HG) promotes neuronal damage and microglial activation by targeting the microRNA-153-3p/alpha-synuclein axis in Parkinson's disease. Bioengineered . 13 (2), 4493-4516 (2022). Thome, A.D., Harms, A.S., Volpicelli-Daley, L.A. & Standaert, D.G. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease. J Neurosci . 36 (8), 2383-2390 (2016). Valera, E. et al. MicroRNA-101 Modulates Autophagy and Oligodendroglial Alpha-Synuclein Accumulation in Multiple System Atrophy. Front Mol Neurosci . 10 (329 (2017). Liu, Q. et al. circ-Pank1 promotes dopaminergic neuron neurodegeneration through modulating miR-7a-5p/α-syn pathway in Parkinson's disease. Cell Death Dis . 13 (5), 477 (2022). Su, Y. et al. MicroRNA-26a/Death-Associated Protein Kinase 1 Signaling Induces Synucleinopathy and Dopaminergic Neuron Degeneration in Parkinson's Disease. Biol Psychiatry . 85 (9), 769-781 (2019). Taghizadeh, E. et al. LncRNAs as putative biomarkers and therapeutic targets for Parkinson's disease. Neurol Sci . 42 (10), 4007-4015 (2021). Fan, Y., Zhao, X., Lu, K. & Cheng, G. LncRNA BDNF-AS promotes autophagy and apoptosis in MPTP-induced Parkinson's disease via ablating microRNA-125b-5p. Brain Res Bull . 157 (119-127 (2020). Qian, C. et al. Downregulated lncRNA-SNHG1 enhances autophagy and prevents cell death through the miR-221/222 /p27/mTOR pathway in Parkinson's disease. Exp Cell Res . 384 (1), 111614 (2019). Statello, L., Guo, C.J., Chen, L.L. & Huarte, M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol . 22 (2), 96-118 (2021). Hu, Y.P. et al. LncRNA-HGBC stabilized by HuR promotes gallbladder cancer progression by regulating miR-502-3p/SET/AKT axis. Mol Cancer . 18 (1), 167 (2019). Bridges, M.C., Daulagala, A.C. & Kourtidis, A. LNCcation: lncRNA localization and function. J Cell Biol . 220 (2) (2021). Zheng, Y.L. et al. LINC01554-Mediated Glucose Metabolism Reprogramming Suppresses Tumorigenicity in Hepatocellular Carcinoma via Downregulating PKM2 Expression and Inhibiting Akt/mTOR Signaling Pathway. Theranostics . 9 (3), 796-810 (2019). Shi, G. et al. Long non-coding RNA LINC00511/miR-150/MMP13 axis promotes breast cancer proliferation, migration and invasion. Biochim Biophys Acta Mol Basis Dis . 1867 (3), 165957 (2021). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4723150","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334874979,"identity":"11c5c2a8-309b-4d8f-8c54-6241b0c562f9","order_by":0,"name":"Rui Duan","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Duan","suffix":""},{"id":334874980,"identity":"a0ef42b2-8b21-4add-94dc-9cb525eebcad","order_by":1,"name":"Liang Shi","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Shi","suffix":""},{"id":334874981,"identity":"f8190c53-ec82-476c-9a18-2bd3563d5292","order_by":2,"name":"Yang Deng","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Deng","suffix":""},{"id":334874982,"identity":"75311609-6de0-4c65-b1fd-08161f3f2aff","order_by":3,"name":"Jiang Wu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Wu","suffix":""},{"id":334874983,"identity":"c81103a3-a271-4e7b-9074-28a1eeec9008","order_by":4,"name":"Shiyao Wang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shiyao","middleName":"","lastName":"Wang","suffix":""},{"id":334874984,"identity":"f39c6221-4f70-444d-94af-2a7cf601b669","order_by":5,"name":"Qiang Peng","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Peng","suffix":""},{"id":334874985,"identity":"d3b13ad5-f78d-4a94-95fe-efb49696d8aa","order_by":6,"name":"Zhongyuan Li","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhongyuan","middleName":"","lastName":"Li","suffix":""},{"id":334874986,"identity":"2200015b-60b3-41ef-b212-a60af2384384","order_by":7,"name":"Zhaohan Xu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhaohan","middleName":"","lastName":"Xu","suffix":""},{"id":334874987,"identity":"bdbe444f-9897-4b45-98b2-0947d79dcd1f","order_by":8,"name":"Feng Wang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Wang","suffix":""},{"id":334874988,"identity":"486bb971-356f-4d08-9b64-024906c5d6cb","order_by":9,"name":"Xue Xue","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Xue","suffix":""},{"id":334874989,"identity":"0bfe2bc8-4212-45a8-9a03-1140eed9beff","order_by":10,"name":"Qing Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYLCCBAYGHn725gMHPhj8k2Njbz9AlBYZyZ5jiQ9nVBww5uM5k0CURTYGN3KMjXnOHEicJ+FggFepvPvhgzce7qjlMThzxkyCt+1OepsE0NYfFdtwajE8k5ZskXjmOI/k8bYyCcm2Z7lt0o0HGHvO3MatpSHHTCKx7RgP35nD2yQM25hz22QOJDAztuHR0v8GooXhRgKIwZzOJpFggFeLvATYlhoegRspxgYHzhxOIKjFQOIZ0C9tB3jAgdxQkWbYBgzkg/j8It+ffPDmz7Y6e1BUHv5jYCMv395+8MGPCjy2HGBgkGBgOIwqegCnepAtDWAtdfjUjIJRMApGwUgHAPchYsOKRcpRAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qing","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-07-11 09:29:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4723150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4723150/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-76058-w","type":"published","date":"2024-11-01T16:20:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62189432,"identity":"b88234a0-49ac-4315-9088-435cd42e32df","added_by":"auto","created_at":"2024-08-10 12:21:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1926159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVE0991 improves behavior disorder and decreased α-syn expression in the hα-syn (A53T) mice. \u003c/strong\u003e(A) Mass spectra of AVE0991 detected with full scan mass spectrometry (MS). MS/MS spectra of precursor ion at m/z 581 from the brain of mice (n=6). (B) For the Rotarod test, the latency of mice to fall off the rod is shown for all groups (n=9). (C) Locomotor activity was assessed with the open field test. The graph shows the movement time (n=9). (D) [18F] FDG PET/CT full body image of mice (n=9). (E) Representative IHC staining images for α-syn in mesencephalon of mice. Scale bar, 50 μm (n=6). Data are shown as the mean ± SD.\u003csup\u003e ****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the Control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 versus the hα-syn (A53T) group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/5cdab151a54d26350f5d1a27.png"},{"id":62190755,"identity":"03b107b5-6b01-4c2b-8628-ba0e7e8ff9b0","added_by":"auto","created_at":"2024-08-10 12:29:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":744184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA HOTAIRM1 induces dopaminergic neurons apoptosis. \u003c/strong\u003e(A) The heatmap represents hierarchical clustering for differentially expressed lncRNAs in right SNpc of each group mice. (B) The expression of HOTAIRM1 in right SNpc from the indicated group were detected by qRT-PCR (n=3). (C) The expression of HOTAIRM1 in primary dopaminergic neurons from the indicated group were detected by qRT-PCR (n=3). (D) The expression of α-syn in primary dopaminergic neurons from the indicated group were detected by Western blot. β-actin served as a loading control (n=3). (E) A bar presenting the quantification of α-syn in indicated group. (F) The apoptosis of primary dopaminergic neurons in indicated group was detected by flow cytometry (n=3). (G) A bar presenting the quantification of apoptosis rate in primary dopaminergic neurons from the indicated group. (H) Western blot to analysis the expression of Bcl2, Bax and cleaved caspase-3 in primary dopaminergic neurons. β-actin served as a loading control (n=3). (I) A bar presenting the quantification of Bcl2 in primary dopaminergic neurons from the indicated group. (J) A bar presenting the quantification of Bax in primary dopaminergic neurons from the indicated group. (K) A bar presenting the quantification of cleaved caspase-3 in primary dopaminergic neurons from the indicated group. Data are shown as the mean ± SD. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the hα-syn (A53T) group, LV-NC group or hα-syn (A53T) + LV-NC group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the sh-NC and hα-syn (A53T) + sh-NC group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/5d95344e1e96a06db6463608.png"},{"id":62190757,"identity":"591e473e-434e-428c-9f7b-c4200bf3ac2f","added_by":"auto","created_at":"2024-08-10 12:29:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":514087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVE0991 inhibits dopaminergic neuron apoptosis induced by lncRNA HOTAIRM1.\u003c/strong\u003e (A) The expression of α-syn in primary dopaminergic neurons from the indicated group were detected by Western blot. β-actin served as a loading control (n=3). (B) A bar presenting the quantification of α-syn in indicated group. (C) The apoptosis of primary dopaminergic neurons in indicated group was detected by flow cytometry (n=3). (D) A bar presenting the quantification of apoptosis rate in primary dopaminergic neurons from the indicated group. (E) Western blot to analysis the expression of Bcl2, Bax and cleaved caspase-3 in primary dopaminergic neurons. β-actin served as a loading control (n=3). (F) A bar presenting the quantification of Bcl2 in primary dopaminergic neurons from the indicated group. (G) A bar presenting the quantification of Bax in primary dopaminergic neurons from the indicated group. (H) A bar presenting the quantification of cleaved caspase-3 in primary dopaminergic neurons from the indicated group. Data are shown as the mean ± SD. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the hα-syn (A53T) + LV-NC group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the hα-syn (A53T) + LV-NC + AVE0991 group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/c48c99cd9f251258e118ab52.png"},{"id":62189436,"identity":"e8c6cbb6-1662-44bd-b3ae-0d6b1cd10790","added_by":"auto","created_at":"2024-08-10 12:21:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1887668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-223-3p binds to HOTAIRM1 as a ceRNA and directly targets α-syn to inhibit apoptosis in dopaminergic neurons.\u003c/strong\u003e (A and B) Possible miR-223-3p binding site on LncRNA HOTAIRM1. (C) Possible miR-223-3p binding site on α-syn mRNA. (D) Representative images of localization of HOTAIRM1 (red) and miR-223-3p (green) in the mice brain slices, and co-stained with TH (pink) for the dopaminergic neurons localization and DAPI (blue) for nuclear localization. Scale bar, 50 μm. (E) qRT-PCR to analysis the miR-223-3p level in primary dopaminergic neurons from the indicated group (n=3). (F) Dual luciferase reporter gene analysis to confirm the binding relationship between HOTAIRM1 and miR-223-3p (n=3). (G) Relative luciferase activity of α-syn wild-type and 3ʹ-UTR mutant structures transfected with miR-223-3p mimics and NC mimic (n=3). (H) qRT-PCR to analysis the miR-223-3p level in primary dopaminergic neurons from the indicated group (n=3). (I) The expression of α-syn in primary dopaminergic neurons from the indicated group were detected by Western blot. β-actin served as a loading control (n=3). (J) A bar presenting the quantification of α-syn in indicated group. (K) The apoptosis of primary dopaminergic neurons in indicated group was detected by flow cytometry (n=3). (L) A bar presenting the quantification of apoptosis rate in primary dopaminergic neurons from the indicated group. (M) Western blot to analysis the expression of Bcl2, Bax and cleaved caspase-3 in primary dopaminergic neurons. β-actin served as a loading control (n=3). (N-P) A bar presenting the quantification of Bcl2, Bax and cleaved caspase-3 respectively in primary dopaminergic neurons from the indicated group. Data are shown as the mean ± SD. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the hα-syn (A53T) + LV-NC group, NC mimic group and hα-syn (A53T) + NC mimic group;\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the syn (A53T) + sh-NC group, NC inhibitor group and hα-syn (A53T) + NC inhibitor group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/1477e2f2e55c8bf7b152675e.png"},{"id":62190756,"identity":"99905132-c8e0-4952-91df-b3b1d9f97314","added_by":"auto","created_at":"2024-08-10 12:29:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":539544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAVE0991 inhibits the apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) by a miR-223-3p-dependent manner.\u003c/strong\u003e (A) The qRT-PCR result of miR-223-3p expression in right SNpc from the indicated group (n=3). (B) The expression of α-syn in primary dopaminergic neurons from the indicated group were detected by Western blot. β-actin served as a loading control (n=3). (C) A bar presenting the quantification of α-syn in indicated group. (D) The apoptosis of primary dopaminergic neurons in indicated group was detected by flow cytometry (n=3). (E) A bar presenting the quantification of apoptosis rate in primary dopaminergic neurons from the indicated group. (F) Western blot to analysis the expression of Bcl2, Bax and cleaved caspase-3 in primary dopaminergic neurons. β-actin served as a loading control (n=3). (G) A bar presenting the quantification of Bcl2 in primary dopaminergic neurons from the indicated group. (H) A bar presenting the quantification of Bax in primary dopaminergic neurons from the indicated group. (I) A bar presenting the quantification of cleaved caspase-3 in primary dopaminergic neurons from the indicated group. Data are shown as the mean ± SD. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 versus the the hα-syn (A53T) group, hα-syn (A53T) + NC inhibitor group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and\u003csup\u003e ###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the hα-syn (A53T) + AVE0991 + NC inhibitor group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/81f6221a6ad365c3939d3c4a.png"},{"id":62189437,"identity":"5064fa8f-0531-47b2-b363-f086b4074ae6","added_by":"auto","created_at":"2024-08-10 12:21:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative level of miR-223-3p of PD patients is significantly correlated with the level of α-syn. \u003c/strong\u003e(A) The relative level of miR-223-3p in blood from PD patients or healthy controls. (B) The level of α-syn in plasma from PD patients or healthy controls. (C) Spearman correlation analysis of the association between miR-223-3p and α-syn expression levels in blood from PD patients or healthy controls. Data are shown as the mean ± SD. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/a6bce224e229c7f0dbd26a37.png"},{"id":68207275,"identity":"825e3686-2aed-4e7c-86c7-954acd655d5f","added_by":"auto","created_at":"2024-11-04 16:36:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7545527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/13f00761-8708-41ff-8d6d-d31c8d9586c3.pdf"},{"id":62189435,"identity":"80822904-eab6-4b2e-9327-4ae81688e924","added_by":"auto","created_at":"2024-08-10 12:21:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":291902,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4723150/v1/6aff7290b3c6635159bc6c6e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"AVE0991 regulates HOTAIRM1/miR-223-3p/α-synuclein to improve behaviour and protect dopaminergic neurons in hu-man α- syn (A53T) overexpressing mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson's disease (PD) is the 2nd prevalent neurodegenerative disease and is expected to impose heavy stresses and economic burdens on society as the population ages \u003csup\u003e1\u003c/sup\u003e. Treatment of PD includes pharmacologic approaches associated with levodopa preparations, nonpharmacologic approaches (such as exercise and physical, occupational, and speech therapies) and deep brain stimulation \u003csup\u003e2, 3\u003c/sup\u003e. However, all above methods only provide symptomatic relief without delaying the progression of the disease \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe renin-angiotensin system (RAS), which is widely distributed in various brain regions, includes the ACE2/Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)/ Mas axis \u003csup\u003e5\u003c/sup\u003e. As found in prior studies, Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) is found in the hippocampus, thalamus and substantia nigra, and has beneficial roles in multiple neurological disorders, including PD \u003csup\u003e6, 7\u003c/sup\u003e. For example, Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) attenuates blood-brain barrier damage following cerebral ischemia-reperfusion by regulating TIMP-1/MMP-9 \u003csup\u003e8\u003c/sup\u003e. Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) decreases inflammation levels and significantly improves cognitive outcomes in traumatic brain injury (TBI) mice \u003csup\u003e9\u003c/sup\u003e. Meanwhile, in 6-hydroxydopamine Parkinsonian rats, Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) could inhibit HMGB-1/RAGE/NF-κB signaling to ameliorate dyskinesia \u003csup\u003e10\u003c/sup\u003e. Notably, Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) exhibits an extremely short half-life (10\u0026ndash;20 s) and must be administered intensively to act in the nervous system \u003csup\u003e11\u003c/sup\u003e. Therefore, we chose AVE0991, the non-peptide analogue of Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), for intraperitoneal administration to conduct our study. Our previous researches have demonstrated that, AVE0991 could reduce neuroinflammation and exerts protective effects in both AD mouse model and mouse model of accelerated aging \u003csup\u003e12, 13\u003c/sup\u003e. However, no study has clarified whether AVE0991 regulates the progression of PD and the specific mechanism.\u003c/p\u003e \u003cp\u003eBased on the genetic characteristics, Parkinson\u0026rsquo;s disease can be divided into familial and sporadic types \u003csup\u003e14\u003c/sup\u003e. Numerous researches have indicated that α-synuclein (α-syn) acts as an important pathogenetic agent in both forms of the disease over the past decades. Intraneuronal proteinaceous cytoplasmic inclusions, which are pathologically characteristic of PD, cause α-syn deposition \u003csup\u003e15\u003c/sup\u003e. Additionally, the A53T mutation of NG_011851 (SNCA) gene encoding hα-syn can lead to autosomal-dominant PD \u003csup\u003e16\u003c/sup\u003e. Our preliminary research shows that Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) decreases α-syn deposition in Parkinson's disease \u003csup\u003e17\u003c/sup\u003e, yet the underlying mechanism is still unclear.\u003c/p\u003e \u003cp\u003eMiRNAs, as a sort of non-coding RNA, which have been recognized to be involved in the developmental process of several neurological diseases \u003csup\u003e18\u003c/sup\u003e. For instance, miRNA-326 was found to diminish tau phosphorylation and neuron apoptosis in Alzheimer's disease by targeting VAV1 \u003csup\u003e19\u003c/sup\u003e. Moreover, miR-96-5p alleviates cerebral ischemia-reperfusion injury in mice by inhibiting pyroptosis via downregulating caspase-1 \u003csup\u003e20\u003c/sup\u003e. In addition, our previous research has found that AVE0991 alleviates neuroinflammation in AD-like mouse brain by regulating miR-223-3p related pathways, above of which also demonstrates that AVE0991 has a strong neuroprotective property. Meanwhile, the levels of plasma miR-223-3p were significantly decreased in PD patients \u003csup\u003e21, 22\u003c/sup\u003e. Accordingly, we inquired into whether AVE0991 could be functional in PD through the regulation of miR-223-3p and its specific mechanism.\u003c/p\u003e \u003cp\u003eSimilarly, another non-coding RNA, lncRNAs, have been also stated that participated in a variety of neurological diseases \u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. Theoretically, lncRNAs could sponge miRNAs as competitive endogenous RNAs (ceRNAs) and thus regulate target protein levels \u003csup\u003e26\u003c/sup\u003e. For example, lncRNA-AK046375 suppressed oxidative stress by up-regulating MT2 expression through the sequestration of miR-491-5p and thus attenuated neurological impairment after TBI \u003csup\u003e27\u003c/sup\u003e. LncRNA BIRF could promote cerebral ischemia tolerance induced by cerebral ischemia preconditioning through sponge miR-330-5p up-regulation of GLT-1 \u003csup\u003e28\u003c/sup\u003e. Additionally, lncHOTAIRM1 (HOXA transcript antisense RNA myeloid-specific 1) was observed to be significantly elevated in circulating leukocytes in PD patients, and overexpression of HOTAIRM1 increases 6-hydroxydopamine-induced apoptosis in SH-SY5Y cells \u003csup\u003e29\u003c/sup\u003e. Nonetheless, it is still unknown about the explicit mechanism of HOTAIRM1 in PD.\u003c/p\u003e \u003cp\u003eIn this work, we addressed whether AVE0991 exerts neuroprotective effects through HOTAIRM1/miR-223-3p to modulate α-syn in PD models in vitro and in vivo. The study also provided novel diagnostic indicators and drug targets for treatment of PD.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the ethical review Committee of Nanjing First Hospital (Number: KY20190509-05), and written informed consent was obtained from all subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6J mice (weight 20-25 g) were received from Vital River Laboratories (Beijing, China). All mice were kept in chambers at controlled ambient temperature and relative humidity with permission to drink and eat freely. The study was permitted by the Animal Care and Use Committee of Nanjing First Hospital (Number:\u0026nbsp;DWSY-22003128). All mice studies were performed in accordance with the ARRIVE guidelines for reporting animal experiments.\u003c/p\u003e\n\u003cp\u003eMice were randomized into three experimental groups: Control group, h\u0026alpha;-syn (A53T) group, h\u0026alpha;-syn (A53T) + 10 mg/kg AVE0991 group. All mice were intraperitoneally injected with sterile saline (0.9%) or AVE0991 (10 mg/kg, MedChemExpress LLC, NJ, USA) once daily for 30 continuous days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic Injection of AAV- H\u0026Alpha;-Syn (A53T) Vector\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice (8 weeks old) were anesthetized with 2% isoflurane in O\u003csub\u003e2\u003c/sub\u003e (RWD Life Science, Shenzhen, China) and then stabilized on a stereotaxic instrument (David Kopf Instruments). AAV-h\u0026alpha;-syn (A53T) (120 nl - 7.0 \u0026times;10\u003csup\u003e12\u003c/sup\u003e vg/ml) was injected at 0.4 \u0026mu;l/min into the right SNpc of mice (AP = \u0026minus;2.9 mm, L = \u0026minus;1.3 mm, DV = \u0026minus;4.5 mm). Notably, the pipette was kept for 5 min post-injection before being slowly withdrawn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC-MS/MS was performed to determine the amount of AVE0991 in brain of mice injected with AVE0991. Brain tissue was homogenized with 500 \u0026mu;L saline, centrifuged and then transferred 100 \u0026mu;L homogenized supernatant to a tube and reconstituted with 500 \u0026mu;L acetonitrile (Sigma-Aldrich, USA). After centrifugation again, 5 \u0026mu;L of supernatant was taken for sampling. MS spectra were collected from m/z 581.72\u0026rarr;308.1. MS/MS profile was obtained with 43 V collision energy. Data were visualized and analyzed using MultiQuant-MD 3.0.3 (AB Sciex, FC, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rotarod test was utilized to evaluate the balance and motor coordination of mice. Mice were kept on a rotating device (IITC Life Science, CA, USA) with the speed adjusted from 5 rpm to 40 rpm for 300 s. Recording of the time each mouse dropped from the rotarod was performed. Testing was performed 3 times per mouse, at least 30 minutes apart.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Field Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen field test was performed to assess the general locomotor activity of mice. Mice were located in the center of an open field square (47 cm\u0026times; 47 cm \u0026times; 40 cm) for 5 min. During this period, a TSE software (TSE Systems, Bad Homburg, Germany) was used to analyze the exercise time of mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePET/CT Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were scanned for [\u003csup\u003e18\u003c/sup\u003eF] FDG PET 30 days after AVE 0991 injection. Briefly, mice were fasted for 6 h and then injected with [\u003csup\u003e18\u003c/sup\u003eF] FDG through the tail vein. 45 minutes later mice were anesthetized with 2.5% isoflurane, fixed, and then subjected to PET scanning acquisition (10 minutes, Inveon, Siemens) as well as CT scanning. Maximum standardized uptake values were computed to estimate [\u003csup\u003e18\u003c/sup\u003eF] FDG uptake content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSN-containing mouse brain sections were deparaffinized, hydrated, antigenically repaired, and permeabilized using 0.5% Triton X-100, subsequently blocked with 5% bovine serum albumin at room temperature, and then incubated with primary antibody \u0026alpha;-syn (1:100, ab212184, Abcam, MA, USA) at 4 \u0026deg;C overnight. On the next day, sections were incubated with biotinylated secondary antibody IgG (1:200, ab64256, Abcam, MA, USA) at room temperature. Sections were stained with diaminobenzidine and restained with hematoxylin, dehydrated and sealed, and images were obtained by a fluorescence microscope (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLncRNA Sequencing and Bioinformatic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the right SNpc tissues of mice using TRIzol reagent (Thermo Fisher Scientific) and its concentration and quality were measured by Qubit fluorometer (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. The quality of data processing of raw reads was checked by FastQC-v0.11.8, and low-quality bases and adaptors contamination were trimmed with Fastp-v0.19.5 if necessary. RNA library preparation was performed according to the manufacturer\u0026rsquo;s instructions, using 1 \u0026mu;g of DNase-treated total RNA as input. The libraries were quantified by using an ABI 9700HT Fast Real-Time PCR system (Thermo Fisher Scientific). Samples were sequenced using the NovaSeq platform (Illumina) to generate 150 bp paired-end reads. STAR-v2.7.7a was performed to map clean reads to the reference genome. Normalization of mapped reads and differential analysis of lncRNAs were performed using DESeq2-v1.10.1. Parameters |log2FC| \u0026ge;1 and P \u0026lt; 0.05 was defined significantly differentially expressed lncRNAs. Sequencing database has been archived in GenBank (GSE244050).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was obtained from tissues or cells using Trizol reagent (Thermo Fisher Scientific). RNA was then reversely transcribed to cDNA with a kit (ABM, Richmond, BC, Canada) following measuring the concentration. Next, it was detected using SYBR-Green method on an ABI7500 type sequence detection system (7500, ABI, USA). U6 and \u0026beta;-actin were used as internal references for miRNA and mRNA, respectively. The specific primer sequences for qRT-PCR were shown in\u003cstrong\u003e\u0026nbsp;Table S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Dopaminergic Neurons Culture and Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFetal mice were dissected from mice at 15 days of gestation and the midbrain tissue was isolated. The tissue was then digested with 0.25% trypsin for 10 minutes at 37\u0026deg;C and the cells were precipitated by filtration and centrifugation. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded in plates pre-coated with poly-L-lysine. After incubation for 4 hours at 37 \u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, the medium was replaced with Neurobasal medium containing 2% B27 and incubation was continued for about 8 days with fluid changes every 2 days. The final concentration of AVE0991 used in the experiments was 1\u0026times;10\u003csup\u003e-6\u003c/sup\u003e M.\u003c/p\u003e\n\u003cp\u003eBriefly, miR-223-3p mimic, miR-223-3p inhibitor, and respective negative controls (NC mimic, NC inhibitor) were acquired from Shanghai GenePharma Company, h\u0026alpha;-synuclein (A53T), lenti-HOTAIRM1, sh-HOTAIRM1 and their lentiviral scramble control shRNA were adopted from Shanghai GeneChem Company. Based on the manufacturer\u0026rsquo;s protocol, transfected them to cells with Lipofectamine 3000. Lentivirus infection was performed by following the instructions, while puromycin was then added to screen for stable cells. The expression of target genes or protein was verified by qPCR or Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted from mouse midbrain tissues or cells using RIPA lysate (Beyotime, Shanghai, China) and protein concentration was quantified by a BCA kit (Thermo Scientific, San Jose, CA, USA). After mixing and heating to denaturation by adding an appropriate amount of loading buffer, the samples were separated by sodium dodecyl sulfate-polyacrylamide gels electrophoresis. Following transfer to the membrane it was blocked in 5% non-fat milk at room temperature. This was followed by overnight incubation at 4 \u0026deg;C with the following primary antibodies: anti-\u0026alpha;-syn (1:1000, ab212184, Abcam, MA, USA), anti-bcl2 (1:1000; ab196495; Abcam, MA, USA), anti-bax (1:1000; #2772; CST, MA, USA), anti-caspase-3(1:1000; #9661; CST, MA, USA) and \u0026beta;-actin (1:1000; #4970; CST, MA, USA). The membranes were washed 3 times and then incubated with corresponding HRP labeled IgG at room temperature. Lastly, strip visualization using an ECL kit (Thermo Fisher Scientific) and quantified by analysis using Image J (National Institutes of Health, Bethesda, Maryland, USA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnexin V/Propidium Iodide (PI) Apoptosis Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptosis rate of the cells was detected by flow cytometry. Cells were collected, centrifuged, washed, and resuspended with 500 \u0026mu;L buffer. In compliance with the instructions of the apoptosis detection kit (KGA107, Keygen,China) the cells were stained for 15 min, and the proportion of apoptotic cells was quantitatively analyzed by flow cytometry (FACSCalibur, BD Biosciences, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescent in Situ Hybridization (FISH)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen sections of brains were subjected to in situ hybridization using the HOTAIRM1 and miR-223-3p probe (RiboBio Co., Ltd, Guangzhou, China) according to the protocol described. After hybridization, the sections were blocked with blocking buffer. Then, the sections were incubated with anti-TH (1:100; #45648, Cell Signaling Technology) overnight at 4 \u0026deg;C, and after washing 3 times in PBS, the sections were incubated with Cy5-labeled goat anti-mouse IgG (1:200; GB27301, Servicebio, Wuhan, China) at room temperature. 4ʹ,6-Diamidino-2-phenylindole staining was completed and the sections were blocked and visualized using a microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual Luciferase Reported Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential sites of \u0026alpha;-syn 3\u0026rsquo;UTR binding to miR-223-3p and assessment of miR-223-3p binding to Lnc HOTAIRM1 were analyzed by online bioinformatics database TargetScan and bioinformatics software RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/). The sequences of luciferase reporter plasmids HOTAIRM1-WT and HOTAIRM1-Mut were constructed and cells were co-transfected with plasmids and NC mimic or miR-223-3p mimic with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were collected after 48 h, lysed and assayed for luciferase activity by the dual luciferase reporting and detection system (Promega, WI, USA). The same procedure was used to determine whether \u0026alpha;-syn and miR-223-3p were bound.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from PD patients and healthy controls admitted to Nanjing First Hospital from 2021 to 2022. No significant differences were found in the age and gender of the participants. The study was approved by the ethical review Committee of Nanjing First Hospital (Number: KY20190509-05). All participants signed a written informed consent before participating in this study.\u003c/p\u003e\n\u003cp\u003eThe upper plasma layer was separated by centrifugation (2,500 g, 15 min) within a short period of time after blood samples were collected and the level of \u0026alpha;-syn in plasma was\u0026nbsp;measured by enzyme-linked immunosorbent assay (ELISA) kit (No. JL12231, Jianglai Biologicals, Shanghai, China). The lower samples were extracted for RNA according with the protocol of the manufacturer\u0026apos;s instructions, and the expression of miR-223-3p was determined by qRT-PCR as described above. Clinical parameters are detailed in \u003cstrong\u003eTable S2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed with the Prism 8.0 (GraphPadSoftware, Inc., La Jolla, USA) software. Differences were analyzed by Student\u0026apos;s t-test for both groups or one-way ANOVA between multiple groups. Correlations analysis of blood samples data was evaluated by the Pearson\u0026rsquo;s method. Data are presented as mean \u0026plusmn; SD of at least three independent experiments. Statistical significance was considered if the \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAVE0991 improves behavior disorder and decreased \u0026alpha;-syn expression in the h\u0026alpha;-syn (A53T) mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAVE0991 was measured in the brain of mice after intraperitoneal injection using LC-MS/MS. The characteristic peak of AVE0991 was detected by full-scan MS signals in the brains of administered mice \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. This suggests that AVE0991 can be penetrate into the brain by intraperitoneal injection. During the rotarod test, mice infected with h\u0026alpha;-syn (A53T) virus spent notably less time on the stick than control mice. However, AVE0991 increased stick retention time in mice infected with h\u0026alpha;-syn (A53T) virus after administration \u003cstrong\u003e(Fig. 1B)\u003c/strong\u003e. Mice were tested for spontaneous locomotion via the open-field test, which showed that mice infected with h\u0026alpha;-syn (A53T) virus had remarkably reduced spontaneous motility compared with control mice. However, AVE0991 increased spontaneous activity in A53T mice after administration of AVE0991 \u003cstrong\u003e(Fig. 1C)\u003c/strong\u003e. Subsequently, whole-body PET/CT scans revealed that, as expected, [\u003csup\u003e18\u003c/sup\u003eF] FDG excretion occurred primarily in the urinary bladder, while the midbrain manifested high levels of [\u003csup\u003e18\u003c/sup\u003eF] FDG uptake, and compared to control mice, [\u003csup\u003e18\u003c/sup\u003eF] FDG uptake in the midbrain region of mice infected with h\u0026alpha;-syn (A53T) virus was conspicuously reduced, while [\u003csup\u003e18\u003c/sup\u003eF]\u0026nbsp;FDG uptake in the midbrain region of mice infected with h\u0026alpha;-syn (A53T) virus was increased after administration of AVE0991 \u003cstrong\u003e(Fig. 1D)\u003c/strong\u003e. Besides, immunohistochemical analysis indicated that \u0026alpha;-syn expression was elevated in the substantia nigra of mice infected with h\u0026alpha;-syn (A53T) virus compared with the control group, whilst AVE0991 administration relatively reduced \u0026alpha;-syn deposition in the substantia nigra of mice infected with h\u0026alpha;-syn (A53T) virus \u003cstrong\u003e(Fig. 1E)\u003c/strong\u003e. These results suggested that AVE0991 ameliorated behavioral deficits and decelerated \u0026alpha;-syn accumulation in the substantia nigra of h\u0026alpha;-syn (A53T) mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLncRNA HOTAIRM1 induces apoptosis in h\u0026alpha;-syn (A53T)-overexpressing dopaminergic neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLncRNAs have been described to be strongly associated with a range of neurological disorders.\u0026nbsp;To investigate whether lncRNAs are involved in AVE0991 therapeutic effects on mice infected with h\u0026alpha;-syn (A53T) virus, we carried out high-throughput lncRNA sequencing of right SNpc tissues in mice infected with h\u0026alpha;-syn (A53T) virus with or without AVE0991 treatment.\u0026nbsp;As illustrated in \u003cstrong\u003eFig. 2A\u003c/strong\u003e, 19 differentially expressed lncRNAs were identified in total after AVE0991 treatment, with corrected thresholds of \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001 and |log2FC| \u0026gt; 1.5.\u0026nbsp;Of those, 12 lncRNAs were prominently up-regulated, whereas the other 7 lncRNAs were notably down-regulated.\u0026nbsp;Our preliminary study shows that elevated levels of HOTAIRM1 expression in the blood of PD patients was elevated\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e. Meanwhile, the HOTAIRM1 levels in right SNpc of mice overexpressing h\u0026alpha;-syn (A53T) after administration of AVE0991 were further confirmed by qRT-PCR\u003cstrong\u003e\u0026nbsp;(Fig. 2B)\u003c/strong\u003e. Then, we further knocked down or overexpressed HOTAIRM1 in primary dopaminergic neurons with h\u0026alpha;-syn (A53T) overexpression\u003cstrong\u003e\u0026nbsp;(Fig. 2C)\u003c/strong\u003e. Western blot findings indicated that \u0026alpha;-syn expression was clearly increased in neurons that overexpressed HOTAIRM1, while \u0026alpha;-syn expression was decreased by knocking down HOTAIRM1 compared with the corresponding control group \u003cstrong\u003e(Fig. 2D\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eE)\u003c/strong\u003e. We determined neuronal apoptosis using flow cytometry. The data suggested that HOTAIRM1 overexpression increased apoptosis in primary dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) compared with the corresponding controls, while knockdown of HOTAIRM1 relatively reduced the apoptosis rate \u003cstrong\u003e(Fig. 2F\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eG)\u003c/strong\u003e. Meanwhile, we further examined several apoptosis-related proteins. Western blot analysis disclosed that compared with the corresponding controls, the expression of Bcl2 was obviously down-regulated and the expression of Bax and cleaved caspase-3 was markedly up-regulated in primary dopaminergic neurons overexpressing HOTAIRM1, while knockdown of HOTAIRM1 upregulated Bcl2 expression and decreased Bax and cleaved caspase-3\u0026nbsp;expression\u003cstrong\u003e\u0026nbsp;(Fig. 2H-K)\u003c/strong\u003e. All above-mentioned findings suggested that lncRNA HOTAIRM1 induced apoptosis in h\u0026alpha;-syn (A53T)-overexpressing dopaminergic neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVE0991 inhibits the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) by a lncRNA HOTAIRM1-manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, to further investigate whether AVE0991 affects the pathological progression of \u0026alpha;-syn via HOTAIRM1, we examined the relative expression of \u0026alpha;-syn in h\u0026alpha;-syn (A53T)-overexpressing primary dopaminergic neurons. Western blot analysis demonstrated that AVE0991 treatment down-regulated \u0026alpha;-syn expression in primary dopaminergic neurons infected with h\u0026alpha;-syn (A53T) virus compared with the corresponding controls, whereas overexpression of HOTAIRM1 reversed the reduction of \u0026alpha;-syn induced by AVE0991 treatment \u003cstrong\u003e(Fig. 3A, B).\u003c/strong\u003e Meanwhile, we probed the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) by flow cytometry. The results showed that AVE0991 treatment reduced apoptosis in primary dopaminergic neurons compared with controls, while overexpression of HOTAIRM1 reversed the protective effect of AVE0991-induced neurons \u003cstrong\u003e(Fig. 3C, D).\u003c/strong\u003e Furthermore, Western blot data illustrated that, compared with controls, AVE0991 treatment in primary dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) showed that Bcl-2 expression was upregulated and Bax expression was downregulated, while cleaved caspase-3 expression was downregulated, while further overexpression of HOTAIRM1 reversed the above results \u003cstrong\u003e(Fig. 3E-H).\u003c/strong\u003e The mentioned results demonstrated that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) through lncRNA HOTAIRM1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-223-3p binds to HOTAIRM1 as ceRNA and directly targets \u0026alpha;-syn to inhibit apoptosis in dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly,\u0026nbsp;we used bioinformatics software RNAhybrid and TargetsScan to identify potential miRNAs that could bind to HOTAIRM1. The analysis showed that the sequence of miR-223-3p is capable of complementary binding to the 3\u0026prime;-UTR sequence of HOTAIRM1 and \u0026alpha;-syn \u003cstrong\u003e(Fig. 4A-C)\u003c/strong\u003e. Next, we performed subcellular localization of several related molecules by FISH assay. The results indicated caspase-3 that in mouse dopaminergic neurons infected with h\u0026alpha;-syn (A53T) virus, HOTAIRM1 was mainly in the neuronal cytoplasm and miR-223-3p was also co-localized with HOTAIRM1 in the cytoplasm \u003cstrong\u003e(Fig. 4D)\u003c/strong\u003e. Thus, we hypothesized that miR-223-3p might bind to HOTAIRM1 as ceRNA, thereby further regulating the expression of target genes after transcription. To validate further the relevance between miR-223-3p and HOTAIRM1, HOTAIRM1 was overexpressed or knocked down in primary dopaminergic neurons infected with h\u0026alpha;-syn (A53T) virus and then detected miR-223-3p levels with qRT-PCR. The outcomes displayed that overexpression of HOTAIRM1 suppressed miR-223-3p levels, while knockdown of HOTAIRM1 increased miR-223-3p levels compared with the corresponding controls \u003cstrong\u003e(Fig. 4E)\u003c/strong\u003e. Subsequently, the test results for the dual luciferase reporter gene assay were met with expectations. miR-223-3p dramatically decreased HOTAIRM1-WT luciferase activity with no reduction in HOTAIRM1-mut activity\u0026nbsp;\u003cstrong\u003e(Fig. 4F)\u003c/strong\u003e. Also, the dual luciferase reporter gene assay exhibited that miR-223-3p greatly decreased \u0026alpha;-syn-WT luciferase activity, yet not \u0026alpha;-syn-MUT activity \u003cstrong\u003e(Fig. 4G),\u003c/strong\u003e which also indicated that \u0026alpha;-syn is a target protein of miR-223-3p. Next, we transfected miR-223-3p mimics or miR-223-3p inhibitors in dopaminergic neurons of mice overexpressing h\u0026alpha;-syn (A53T) \u003cstrong\u003e(Fig. 4H)\u003c/strong\u003e. Western blot results identified that \u0026alpha;-syn expression was declined in neurons overexpressing miR-223-3p compared to the corresponding controls, while miR-223-3p knockdown relatively increased \u0026alpha;-syn expression \u003cstrong\u003e(Fig. 4I, J)\u003c/strong\u003e. We examined the apoptosis of neurons by flow cytometry. It was found that miR-223-3p mimics transfection reduced apoptosis induced by infection with h\u0026alpha;-syn (A53T) virus compared with the corresponding controls, while miR-223-3p inhibitors transfection relatively exacerbated the rate of apoptosis \u003cstrong\u003e(Fig. 4K, L)\u003c/strong\u003e. Meanwhile, we further examined several apoptosis-related proteins. Western blot analysis showed that, compared with the corresponding control group, the expression of Bcl2 was up-regulated in neurons overexpressing miR-223-3p, the expression of Bax was down-regulated, and cleaved caspase-3 expression was down-regulated, whereas knockdown of miR-223-3p was reversed \u003cstrong\u003e(Fig. 4M-P)\u003c/strong\u003e. To summarize,\u0026nbsp;these results prove that miR-223-3p acts as a ceRNA binding to HOTAIRM1 and directly targets \u0026alpha;-syn to inhibit the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVE0991 inhibits the apoptosis of dopaminergic neurons overexpressing A53T \u0026alpha;-syn by up-regulating miR-223-3p\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further explored whether AVE0991 inhibited \u0026alpha;-syn expression through miR-223-3p. qRT-PCR results displayed that compared with the control group, treatment\u0026nbsp;of AVE0991 increased the levels of miR-223-3p in right SNpc of mice overexpressing h\u0026alpha;-syn (A53T) \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e. Western blot demonstrated that AVE0991 treatment decreased \u0026alpha;-syn expression in primary dopaminergic neurons of overexpressed h\u0026alpha;-syn (A53T) mice compared with the corresponding control group, while transfection of miR-223-3p inhibitors reversed the decrease of \u0026alpha;-syn elicited by AVE0991 treatment \u003cstrong\u003e(Fig. 5B, C)\u003c/strong\u003e. Meanwhile, the apoptosis of primary dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) was detected by flow cytometry. Results showed that treatment of AVE0991 reduced neuronal apoptosis, while transfection of miR-223-3p inhibitors reversed this effect of AVE0991 \u003cstrong\u003e(Fig. 5D, E)\u003c/strong\u003e. Western blot analysis indicated that, compared with the control group, following administration of AVE0991, primary dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) had noticeably up-regulated expression of Bcl2, down-regulated expression of Bax and cleaved caspase-3. Further miR-223-3p inhibitors transfection reversed the above results \u003cstrong\u003e(Fig. 5F-I)\u003c/strong\u003e. These findings suggested that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) through upregulation of miR-223-3p expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative plasma levels of miR-223-3p in PD patients are significantly correlated with levels of \u0026alpha;-syn\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, with the purpose of further clarify the clinical significance of miR-223-3p and \u0026alpha;-syn as biomarkers of PD, plasma was collected from PD patients and healthy individuals and then measured the levels of miR-223-3p and \u0026alpha;-syn.\u0026nbsp;Analysis of qRT-PCR resulted in decreased expression of miR-223-3p in the plasma of PD patients compared to healthy controls \u003cstrong\u003e(Fig. 6A)\u003c/strong\u003e. Meanwhile, compared to healthy controls, plasma \u0026alpha;-syn expression in PD patients was relatively elevated \u003cstrong\u003e(Fig. 6B)\u003c/strong\u003e. The above results were similar to the trend of miR-223-3p and \u0026alpha;-syn expression levels in animal and cellular models. Then, we evaluated the correlation between miR-223-3p and \u0026alpha;-syn levels in plasma of PD patients, finding that there was a significant negative correlation between the plasma levels of miR-223-3p and \u0026alpha;-syn expression in PD patients \u003cstrong\u003e(Fig. 6C)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePD, one of the most common synucleinopathies, are pathologically characterized by aggregated \u0026alpha;-synuclein riboproteins forming neurons or glial intracytoplasmic inclusions\u0026nbsp;\u003csup\u003e31, 32\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIt is identified that the SNCA gene was discovered to contain a single point mutation leading to a non-synonymous amino acid substitution in p.A53T, resulting in familial early-onset PD\u0026nbsp;\u003csup\u003e33, 34\u003c/sup\u003e.\u0026nbsp;These also suggest that lowering the expression of \u0026alpha;-syn may be an effective therapeutic option for Parkinson\u0026apos;s disease-like disorders. Therefore, we used a mouse model in which PD-like pathology was evoked by overexpression of human \u0026alpha;-syn (A53T)\u0026nbsp;in dopaminergic neurons in this study, which can help understand progression of PD. Our results demonstrate that mice infected with h\u0026alpha;-syn (A53T) virus exhibited the basic characteristics of PD, including significantly reduced time spent rotating rods and decreased spontaneous activity, compared with control mice. Moreover, \u0026alpha;-syn accumulation increased in the substantia nigra of mice infected with h\u0026alpha;-syn (A53T) virus. In addition to \u0026alpha;-syn deposition, loss of dopaminergic neurons in the nigra, especially in the ventral-lateral portion, is also a pathological signature of PD\u0026nbsp;\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;Therefore, we also constructed an in vitro model of mouse dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) for further study.\u003c/p\u003e\n\u003cp\u003eSince decades ago, the Ang-(1-7) non-peptide analogue AVE0991 was already found to play an influential task in diseases of various systems. For example, it has been reported that AVE0991 has a cardioprotective effect under hyperglycemia conditions\u0026nbsp;\u003csup\u003e36\u003c/sup\u003e. Murphy et al. found that AVE0991 has certain antitumorigenic and anticachectic actions, suggesting it may be useful in the treatment of cancer or other severe muscular dystrophy conditions\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. Meanwhile, AVE0991 was proven that can ameliorate arthritic damage by diminishing neutrophil infiltration and cytokine generation\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e.\u0026nbsp;In the study, our findings demonstrated that AVE0991 can alleviate the behavioural disorder of h\u0026alpha;-syn (A53T) overexpressing mice, and at the same time, AVE0991 can relatively reduce the apoptosis of mice dopaminergic neurons overexpressed h\u0026alpha;-syn (A53T).\u003c/p\u003e\n\u003cp\u003eEmerging evidence suggested that\u0026nbsp;miRNAs contribute to the pathogenesis of PD\u003csup\u003e26\u003c/sup\u003e. A host of miRNAs are differentially expressed in PD and impact the process, including miR-153-3p\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003csup\u003e39\u003c/sup\u003e, miR-155\u0026nbsp;\u003csup\u003e40\u003c/sup\u003e, and miR-101\u0026nbsp;\u003csup\u003e41\u003c/sup\u003e, etc. Of special note, it was found that miR-7a-5p can target and modulate the \u0026alpha;-syn\u0026nbsp;expression, the marker of PD, in mouse dopaminergic neurons\u0026nbsp;\u003csup\u003e42\u003c/sup\u003e. Su et al. testified that inhibition of miR-26a expression leads to dopaminergic neuronal cell death, and motor deficits in normal mice\u0026nbsp;\u003csup\u003e43\u003c/sup\u003e. It is worth emphasizing that in the present research, we verified miR-223-3p directly targets \u0026alpha;-syn for inhibiting apoptosis in dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T). AVE0991 restrained apoptosis in dopaminergic neurons overexpressing A53T \u0026alpha;-syn by up-regulating miR-223-3p.\u003c/p\u003e\n\u003cp\u003eA growing body of research has suggested that lncRNAs perform an essential job for neurodegenerative conditions, including PD\u0026nbsp;\u003csup\u003e44\u003c/sup\u003e. Commonly, in neurological diseases, lncRNA can affect the target RNA levels, and a break in the balance between lncRNA and target RNAs can cause changes in neuronal viability and induces the survival or death of neurons. For example, it is reported that elevated expression of lncRNA BDNF-AS promotes MPTP-induced apoptosis and autophagy in PD model mice\u0026nbsp;\u003csup\u003e45\u003c/sup\u003e. It has been observed that repression of lncRNA UCA1 expression reduces MPP\u003csup\u003e+\u003c/sup\u003e-induced inflammation and oxidative stress in PD cells, thereby alleviating dopaminergic neuronal damage\u0026nbsp;\u003csup\u003e46\u003c/sup\u003e. In light of the above findings, we then explored the participation of lncRNAs in regulation of the progression of PD after administration of AVE0991 in mice with h\u0026alpha;-syn\u0026nbsp;(A53T) overexpression. Sequencing outcomes combined with qRT-PCR data showed that in dopaminergic neurons of AVE0991-treated h\u0026alpha;-syn (A53T) overexpressing mice, the lncRNA HOTAIRM1 was down-regulated. In vitro, knocking down HOTAIRM1 decreased \u0026alpha;-syn expression while overexpression of HOTAIRM1 reversed the \u0026alpha;-syn reduction induced by AVE0991 administration. This observation indicated that AVE0991 inhibited the apoptosis of dopaminergic neurons overexpressing h\u0026alpha;-syn (A53T) via a HOTAIRM1-dependent manner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMore and more reports have indicated that the subcellular localization of lncRNA may also affect their biological functions\u0026nbsp;\u003csup\u003e47, 48\u003c/sup\u003e.\u0026nbsp;Numerous\u0026nbsp;lncRNA are distributed in cytoplasm and nucleus, but their distribution is different\u0026nbsp;\u003csup\u003e49\u003c/sup\u003e. In general, lncRNA widely distributed in the cytoplasm mainly affect cell signalling cascades and regulate the stability or translation process of their target mRNA, while lncRNA in the nucleus are involved in RNA processing, transcriptional regulation, and chromatin interactions\u0026nbsp;\u003csup\u003e50, 51\u003c/sup\u003e. Here, FISH testing revealed that HOTAIRM1 was mainly located in the cytoplasm of mouse dopaminergic neurons and co-localized with miR-223-3p in the cytoplasm, suggesting that HOTAIRM1 may have the function of competitively binding to miRNA. Bioinformatics prediction and luciferase reporter gene detection showed that HOTAIRM1 interacts with miR-223-3p. In addition, HOTAIRM1 down-regulates the miR-223-3p expression. Based on the above data, we speculated that miR-223-3p can competitively bind HOTAIRM1 and directly act on the downstream target \u0026alpha;-syn to exert vital effects in the cytoplasm of dopaminergic neurons.\u003c/p\u003e\n\u003cp\u003eOur present study shows that the angiotensin-(1\u0026ndash;7) analogue AVE0991 targeted the HOTAIRM1/miR-223-3p axis to degrade \u0026alpha;-syn in mouse model with PD-like pathology is induced by overexpression of human \u0026alpha;-syn (A53T), and showed neuroprotection in vitro. Here, the study highlights the valuable neuropharmacological benefits of AVE0991 in the course of PD disease. Furthermore, this study supports essential evidence for PD treatment strategies targeting \u0026alpha;-syn. These findings expand our understanding that the angiotensin-(1\u0026ndash;7) analogue AVE0991 has a strong potential for PD therapy and provides support for the discovery of therapeutic approaches for PD and other related synucleinopathies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRui Duan designed and performed most experiments, analyzed data, and prepared the manuscript; Liang Shi and Yang Deng performed experiments and analyzed the data. Jiang Wu and Shiyao Wang helped with the mice model construction. Qiang Peng, Zhongyuan Li and Zhaohan Xu performed experiments; Feng Wang, Xue Xue, and Qing Gao directed the study and prepared the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2406900), the National Natural Science Foundation of China (82301609), China Postdoctoral Science Foundation (2022M711666), China Postdoctoral Science Foundation (2023M741764), Natural Science Foundation of Jiangsu Province (BK20220196), the Medical Science and Technology Program of Nanjing (JQX22006), the International Joint Research and Development Project of Nanjing (202201030), the International Joint Research and Development Project of Nanjing (202308005) and Xinghuo Talent Program of Nanjing First Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ede Lau, L.M. \u0026amp; Breteler, M.M. 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AVE0991, a non-peptide analogue of Ang-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), by which the progression of PD has been discovered to be ameliorated, but the specific mechanism whereby AVE0991 modulates the progression of PD re-mains unclear. The mice overexpressing of human α-syn (A53T) were established to simulate PD pathology, and we also constructed an in vitro model of mouse dopaminergic neurons overexpressing hα-syn (A53T). The [\u003csup\u003e18\u003c/sup\u003eF] FDG-PET/CT method was also employed to assess FDG uptake in human α-syn (A53T) overexpressing mice. Levels of lnc HOTAIRM1, miR-223-3p were detected via qRT-PCR. Flow cytometry was deployed to assay cell apoptosis. Here, we found that AVE0991 improved behaviour disorder and decreased α-syn expression in the substantia nigra in mice with Parkinson's disease. AVE0991 inhibited apoptosis of dopaminergic neurons overexpressing hα-syn (A53T) by lncRNA HOTAIRM1. MiR-223-3p binds to HOTAIRM1 as a ceRNA and directly targets α-syn. Our present study shows that the angiotensin-(\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) analogue AVE0991 targeted at the HOTAIRM1/miR-223-3p axis to degrade α-synuclein in PD mice, and showed neuroprotection in vitro.\u003c/p\u003e","manuscriptTitle":"AVE0991 regulates HOTAIRM1/miR-223-3p/α-synuclein to improve behaviour and protect dopaminergic neurons in hu-man α- syn (A53T) overexpressing mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-10 12:21:49","doi":"10.21203/rs.3.rs-4723150/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-30T09:00:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-29T13:09:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293163625388013198892044720943188390173","date":"2024-08-14T14:21:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-01T17:11:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79074833676084939346155172740361062082","date":"2024-07-24T12:06:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-23T13:48:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-23T03:23:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-11T21:23:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T11:30:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-11T09:26:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89f877f9-a9a8-4de4-b622-c25b2079bf3b","owner":[],"postedDate":"August 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35465031,"name":"Biological sciences/Cell biology"},{"id":35465032,"name":"Biological sciences/Molecular biology"},{"id":35465033,"name":"Biological sciences/Neuroscience"},{"id":35465034,"name":"Health sciences/Diseases"},{"id":35465035,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2024-11-04T16:29:40+00:00","versionOfRecord":{"articleIdentity":"rs-4723150","link":"https://doi.org/10.1038/s41598-024-76058-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-01 16:20:38","publishedOnDateReadable":"November 1st, 2024"},"versionCreatedAt":"2024-08-10 12:21:49","video":"","vorDoi":"10.1038/s41598-024-76058-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-76058-w","workflowStages":[]},"version":"v1","identity":"rs-4723150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4723150","identity":"rs-4723150","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}