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Repurposing the antiplatelet drug prasugrel for Parkinson’s disease: Evidence of neuroprotective effects and proteomic profiles

ABSTRACT: Chronic neuroinflammation is a hallmark pathological feature of Parkinson’s disease (PD) that contributes to progressive neuronal death. Current medications are largely palliative, underscoring the need for alternative therapeutic strategies. Drug repurposing offers a cost-effective and time-efficient approach by leveraging established safety profiles. Here, we screened the U.S. Food and Drug Administration–approved drug library using two in vitro PD models: 1-methyl-4-phenylpyridinium (MPP+)-induced primary cortical neurons and lipopolysaccharide (LPS)-induced BV2 microglial cells. Proteomic alterations following prasugrel treatment were profiled using Ingenuity Pathway Analysis. Neuroprotective effects were validated by biochemical assays and further confirmed in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We found that prasugrel, an antiplatelet drug used for acute coronary syndrome, exhibited significant neuroprotective and anti-inflammatory effects. It reduced the expression of apoptosis- and inflammation-related proteins, inhibited mitogen-activated protein kinase (MAPK)–mediated neuronal death and suppressed nuclear factor kappa B (NF-κB)-dependent inflammatory signaling. In vivo, prasugrel administration preserved dopaminergic neurons and improved motor performance in MPTP-treated mice. These findings demonstrate that prasugrel mitigates neuronal apoptosis and microglial activation through modulation of MAPK and NF-κB signaling pathways, supporting its development as a repurposed therapeutic candidate for PD.

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ORGANISM(S): Rattus Norvegicus (rat) Mus Musculus (mouse)

TISSUE(S): Cerebral Cortex Neuron, Primary Cell, Cell Culture

DISEASE(S): Parkinson's Disease

SUBMITTER: Eunji Cho

LAB HEAD: Jong Hyuk Yoon

PROVIDER: PXD076315 | Pride | 2026-07-16

REPOSITORIES: Pride

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Project description:Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. U2OS cells were grown in DMEM supplemented with 10% FCS, 100 U/l penicillin, and 100 μg/l streptomycin at 37 °C in 10% CO2 and passaged at ~80% confluence. U2OS cells expressing LAP1-tagged MRFAP1 were grown in the same medium but with the addition of 150 μg/ml hygromycin B and 15 μg/ml blasticidine HCl. The cells for each condition were harvested separately via trypsinization, washed in PBS, and lysed in IP buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 7.4, 10% glycerol, 150 mm NaCl, complete protease inhibitor mixture (Roche Applied Science), PhosStop, 50 mm N-ethylmaleimide). The lysates were sonicated for 10 s at 10% power (three times in total) and then centrifuged for 10 min at 17,000g at 4 °C. Equal protein amounts of each sample were then combined with GFP-trap agarose beads from ChromoTek (Martinsried, Germany) that had been washed once in IP buffer (40 μl of 50% GFP-trap bead slurry per IP) and incubated for 2 h at 4 °C with rotation. The beads were then washed three times with IP buffer via centrifugation at 2,000g for 2 min at 4 °C.

Project description:The two-component system (TCS) is a specific regulatory system in bacteria and plays an important role in sensing and adapting to the environment. In this study, we evaluated the roles of TCSs in the major cariogenic pathogen Streptococcus mutans in resistance to several types of bacteriocin. In a comprehensive analysis using individual TCS mutants, we found that two novel TCSs were associated with resistance against distinct lantibiotics nisin A (class I type A[I]) and nukacin ISK-1(class I type A[II]). One TCS, SMU.659-660 (designated as NsrRS), was related to resistance against nisin A produced by Lactococcus lactis ATCC 11454. The other TCS, SMU.1146-1145 (designated as LcrRS), was related to resistance against nukacin ISK-1 produced by Staphylococcus warneri ISK-1. NsrRS induced the expression of SMU.658 (designated as NsrX), which constitutes an operon with nsrRS, in response to nisin A. Inactivation of nsrX increased susceptibility to nisin A. Additionally, NsrX expression in S. mutans increased the binding affinity to nisin A compared to a no-expression strain. LcrRS induced the expression of SMU.1148-50 (lctFEG), which encodes an ABC transporter and is located upstream of lcrRS, in response to nukacin ISK-1. Inactivation of lctFEG significantly increased susceptibility to nukacin ISK-1. Electrophoretic mobility shift assay analysis revealed that NsrR and LcrR bound directly to regions upstream of nsrX and lctFEG, respectively. This is the first report that two distinct TCSs, NsrRS and LcrRS, are independently involved in resistance to nisin A and nukacin ISK-1 in S. mutans. Total of 14 samples were analyzed. Total RNA from each test strain and control described below were labeled with Alexa FluorÂ® 555 and Alexa FluorÂ® 647, respectively, and were cohybridized on a single array. Labeling and hybridization were performed once or twice independently. UA159 wild type as control vs. UA159 with nisinA or nukacin ISK-1, UA159 wild type with nisinA or nukacin ISK-1 vs. nsrRS or lcrRS deletion mutant in UA159 with nisinA or nukacin ISK-1.

Project description:Recent evidences have demonstrated phosphatidylinositol3-kinase (PI3-K)/Akt signaling pathway participates in cell growth, cell survival, and adipocyte differentiation in vitro. However, the in vivo evidence to support Akt function on adipogenesis is limited. To achieve this goal, we generated the stable zebrafish transgenics of Tg(krt4:myrAkt1)cy18 carrying human constitutive active form of Akt1 (myrAkt1) that driven by a skin-specific krt4 promoter. The Tg(krt4:myrAkt1)cy18 display severely skin hypertrophy at embryonic stages, and support Akt1 function as a key gene on cell size control. When transgenics reached sexual maturation, they display obese phenotype due to adipocyte hypertrophy and up-regulation of adipogenesis-related genes. Collectively, our findings provided a direct evidence to support Akt play provital roles on cell size control as well as adipogenesis in vivo. In addition, the obese zebrafish line of Tg(krt4:myrAkt1)cy18 provide a new platform to study obesity and its related chronic diseases mechanism in vivo. The AB strain of Zebrafish (Danio rerio) were obtained from ZIRC (http://zebrafish.org/zirc/home/guide.php), and kept in the stock of Chung Yuan Christian University. Zebrafish were raised in a local tap water at 28.0±0.5℃ and under a constant photoperiodism of 14 hour light cycle/10 hour dark cycle. Zebrafish embryos were collected in 10 cm diameter Petri dishes containing 20 mL fish water after spawning and raised at 28.0±0.5℃. To prevent the disease of Zebrafish embryo, few drops methylen blue were added into fish water. At 5 to 7 dpf, larvae were transferred to 10 liter tanks containing 8.0 liter of fish water and fed with living Paramecium. After 14 dpf, larvae were feed with living artemia (OSI, USA) until adulthood. We dissected two tail tissues (as a pooled sample) from six month-old wild-type and Tg(krt4:myrAkt1)cy18 (n=5), and homogenized in Trizol reagent (Ambion) to isolate total RNA according to the manufacture instruction. The RNA was treated with DNase I at 25℃ for 10 minutes to remove DNA contamination and then cleanup by RNase-free spin columns (Qiagen). The total RNA concentration was determined by spectrophotometry (ND-1000 ; NanoDrop Technol, Wilmington, DE), and the RNA quality was checked by running electrophoresis in RNA-denatured gels. All total RNA were stored at -20℃. For real-time quantitative RT-PCR, 1μg of total RNA were reverse-transcribed with reverse transcriptase and the excess RNA were digested E.coli RNase H to enhance the cDNA purity. The commercial Zebrafish 14K oligo microarray chip was obtained from Institute of Cellular and Organismic Biology at Academia Sinica and contained 14067 oligonucleotides represent 9666 unique genes with a redundancy of 31%. The detail oligonucleotide description can be obtained from Ocimun Biosolution (http://www.ocimumbio.com/web/default.asp). We used SuperScriptTM Indirect cDNA Labeling System kit (Invitrogen) to generate the fluorescently labeled probes. Total RNA isolated from WT and Tg(krt4:myrAkt1)cy18 were reversed transcribed into cDNA and coupling for Alexa Fluor 555 and Alexa Fluor 647 fluorescent dyes (Invitrogen), respectively. Before hybridization, the Zebrafish microarray chips were pretreated with 1% bovine serum albumin, 4X SCC, and 1% sodium dodecylsulfate (SDS) for 45min at 42℃, and then hybridized in SlideHybTM buffer (Ambion) for overnight at 42℃. After hybridization, chips were washed with 2X SSC and 0.5% SDS for 15min at 25℃ and finally with 0.5X SSC and 0.5% SDS for 15min at 25℃. The fluorescence intensities of Alexa Fluor 555 and Alexa Fluor 647 targets were scanned by Genepix scanner (Molecular Devices, Sunnyvale, CA, USA) and the acquired date were analyzed by Genepix and Genespring software (Aglient Technologies, Foster City, CA, USA).

Project description:Inferring the heritability of gene expression is one of the main areas of the field of genetical genomics. With the possibility to treat the abundances of gene transcripts as a suite of quantitative traits, genetical genomics can make an extensive use of the microarray technology. Here we extended a major method for estimating the heritability of a quantitative trait, single parent-offspring regression, to assess the heritability of the expression of genes with two-channel microarrays. In a series of maternal parent-offspring pairs of Interior spruce (Picea glauca x engelmannii, our focus in the outer stem tissues is the expression of defense-related genes, the heritability of which can affect fitness and necessary for evolution by natural selection. Parent-offspring pairs of Interior spruce planted as a part of Tree Breeding and Improvement Program of the Forest Genetics Section, British Columbia Ministry of Forests and Range, Prince George, BC, Canada (http://www.for.gov.bc.ca/hre/forgen/interior/spruce.htm#1) were used. A total of 30 trees, comprising 15 parent-offpsring pairs were sampled. These include15 offsprings and 15 maternal parents (grafts), which were planted within ~ 1Km range west (122º 42’ 43’’W, 53º 45’41’’N) of the offsprings, hence grown in a relatively similar environment. The bark and the attached phloem were separated from inner layers in the mid-morning hours of June 25, 2008, flash-frozen in liquid nitrogen, and transferred into separate containers. A chain design was decided for gene expression profiling (a total of 45 slides) using the Treenomix third generation cDNA microarray platform (GEO accession #: GPL5423). Array350 kit (Genisphere, Hatfield, USA) was chosen for the microarray hybridizations. The slides were scanned with a ProScanArray scanner (PerkinElmer, Downers Grove, IL, USA), and the scanned TIF images were processed by ImaGene software (BioDiscovery, Inc., El Segundo, CA, USA) to quantify spot signals. Normalization was done between arrays using variance stabilizing (VSN) method for ratio based analysis of the expression data.

			Action	DRS
	BV2_L1.raw	Raw
	BV2_L2.raw	Raw
	BV2_L3.raw	Raw
	BV2_LE11_1.raw	Raw
	BV2_LE11_2.raw	Raw

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Repurposing the antiplatelet drug prasugrel for Parkinson’s disease: Evidence of neuroprotective effects and proteomic profiles

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