Gastrodin inhibits the formation of ataxin-3 aggregates by regulating the level of ERK1/2/P38 proteins

Gastrodin inhibits the formation of ataxin-3 aggregates by regulating the level of ERK1/2/P38 proteins | 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 Research Article Gastrodin inhibits the formation of ataxin-3 aggregates by regulating the level of ERK1/2/P38 proteins Zijian Wang, Xunhao Xiao, Min Wang, Ruitong Cheng, Chan Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7147978/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted 6 You are reading this latest preprint version Abstract Background: Spinocerebellar ataxia type 3 (SCA3/Machado-Joseph disease), an incurable autosomal dominant neurodegenerative disorder, is caused by cytotoxic aggregation of polyglutamine-expanded ataxin-3 protein. Novel therapeutic strategies targeting its pathogenesis are urgently needed. Purpose: Given gastrodin's established antioxidative and neuroprotective properties, this study investigated its therapeutic potential against SCA3 pathogenesis. Methods: Three distinct cell models including parental HEK293T, ataxin-3-15Q (physiologic), and ataxin-3-77Q (pathogenic) were employed to assess gastrodin cytotoxicity, quantify insoluble aggregate formation and measure soluble ataxin-3 levels. Mechanistic studies included antioxidant capacity assays, human phosphokinase array profiling (37 kinases) and western blot validation of MAPK pathway components. Results: Gastrodin treatment showed no cytotoxicity, significantly suppressed ataxin-3-77Q aggregate accumulation (p<0.01), increased soluble ataxin-3 levels, enhanced cellular antioxidant capacity and selectively downregulated ERK1/2 and p38 proteins in MAPK pathways. Conclusion: We provide first evidence that gastrodin mitigates polyQ-mediated proteotoxicity by reducing ataxin-3 aggregation through suppression of the ERK1/2-p38 signaling axis, revealing a novel mechanistic basis for SCA3 therapeutic development. Gastrodin SCA3 Ataxin-3 aggregation ERK/p38 signaling Neuroprotection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights 1. Gastrodin's safety profile was demonstrated in SCA3 cellular models at concentrations up to 100 μM without causing cytotoxicity. 2. Gastrodin significantly reduced the formation of polyQ-expanded ataxin-3 aggregates. 3. There was a dose-dependent increase in soluble ataxin-3 levels by gastrodin. 4. Gastrodin was found to attenuate SCA3 proteotoxicity by simultaneously decreasing the total protein levels of ERK1/2 and p38. Introduction Spinocerebellar ataxia type 3 (SCA3/Machado-Joseph disease), the most prevalent autosomal-dominant inherited ataxia globally [ 1 – 2 ], arises from abnormal polyglutamine (polyQ) expansion in ataxin-3. This mutation triggers cytotoxic protein aggregation-a hallmark of polyQ disorders - where misfolded ataxin-3 forms insoluble inclusions that sequester vital cellular components and drive neurodegeneration [ 3 – 4 ]. Critically, no disease-modifying therapies currently exist to halt SCA3 progression [ 5 ]. Pathogenic synergy between proteotoxicity and oxidative stress underpins SCA3 pathogenesis. PolyQ-expanded ataxin-3 directly impairs proteostasis by overwhelming autophagy and ubiquitin-proteasome systems [ 6 ]. Concurrently, it induces mitochondrial dysfunction and redox imbalance, evidenced by elevated oxidative stress markers and diminished antioxidant defenses in patients and models [ 7 ]. This dual insult creates a vicious cycle: oxidative stress accelerates aggregate formation, while aggregates further amplify oxidative damage. Existing therapeutic strategies targeting aggregation (e.g., autophagy enhancers, proteasome activators) or oxidative stress show preclinical promise but face translational barriers including safety concerns and poor blood-brain barrier (BBB) penetration [ 8 ]. Repurposing phytochemicals with established neuroprotective profiles offers a pragmatic alternative [ 9 ]. Gastrodin, the primary bioactive compound from Gastrodia elata Blume, emerges as a compelling candidate due to its multimodal neuroprotection, including demonstrated antioxidant, anti-aggregation, and anti-apoptotic effects in Alzheimer's and Parkinson's models [ 10 – 12 ]; its favorable pharmacokinetics, including proven blood-brain barrier permeability with minimal side effects [ 13 ]; and its unmet potential, never before evaluated in polyQ disorders. Here, we bridge this gap by investigating gastrodin's efficacy against SCA3 proteotoxicity. Using HEK293T-based models expressing physiologic (15Q) or pathogenic (77Q) ataxin-3, we assessed its effects on cell viability, aggregate burden, and soluble ataxin-3 levels, mapped kinase signaling alterations via phosphoproteomic screening, mechanistically dissected its regulation of the ERK1/2-p38 axis - a convergence point for oxidative stress and proteostasis disruption. Results Gastrodin at concentrations ranging from 5 µM to 100 µM is safe in HEK293T cells overexpressing ataxin-3 To investigate the effect of gastrodin on cellular toxicity in SCA3 cell models, two methods, the CCK-8 method and the MTT assay, were used to assess cell proliferation and cell toxicity. CCK-8 assays revealed that gastrodin at concentrations of 5-100 µM did not have cytotoxic effects on cells after 2, 4, 6, 24, or 48 hours (Fig. 1 A-E). The MTT results were consistent with the CCK-8 assay results (Fig. 1 F). These findings indicate that gastrodin is considered safe for use in these SCA3 cell models, with concentrations ranging from 5-100 µM. Gastrodin reduces polyQ-expanded ataxin-3 aggregates To explore the effect of gastrodin on attenuating the aggregates formed by expanded ataxin-3 in a cell model of SCA3, a filter trap assay was employed, and a cell fractionation analysis was performed. Based on the cytotoxicity results and reports in the literature, we selected a gastrodin concentration of 10 µM or 50 µM, which has a protective effect on other neurodegenerative diseases. Filter trap assessment revealed that, compared with DMSO treatment, gastrodin treatment significantly decreased the number of aggregates formed by expanded ataxin-3 (Fig. 2 A). To analyze the population of aggregates decreased by gastrodin, we fractionated the aggregated ataxin-3 using a fractionation assay. Consistently, treatment with gastrodin decreased the amount of insoluble expanded ataxin-3 aggregates observed in formic acid (FA)-treated cells, which is an SDS-insoluble protein that is harmful to cells. Moreover, after gastrodin treatment, the levels of the Triton X-100-soluble and SDS-soluble ataxin-3 proteins increased, indicating that the degraded aggregates formed soluble proteins (Fig. 2 B). Further analyses were conducted to investigate the effect of gastrodin on the total level of soluble ataxin-3 protein. Western blot analysis revealed that 10 or 50 µM gastrodin treatment for 4 or 24 hours significantly increased soluble expanded ataxin-3 levels, while exerting minimal effects on normal ataxin-3 (Fig. 2 C.D). These findings suggest that gastrodin effectively prevents aggregate formation in expanded ataxin-3-expressing HEK293T cells. Gastrodin plays a protective role through the ERK1/2/P38 signaling pathway. To investigate the mechanism underlying the inhibitory effect of gastrodin on aggregates, we first used the total antioxidant capacity assay to assess the antioxidative ability of gastrodin in three cells models. Consistent with previous reports, compared with DMSO treatment, 10 µM gastrodin treatment increased the antioxidative activity in HEK293T cells, 10 µM and 50 µM gastrodin treatment showed an increased the tendency of the antioxidative activity in ataxin-3-15Q-expressing HEK293T cells (Fig. 3 A, B). However, gastrodin did not exhibit antioxidative activity in ataxin-3-77Q-expressing HEK293T cells (Fig. 3 C). These data demonstrated that gastrodin has the antioxidative activity, however, may also play a protective role in other signaling pathways in SCA3. We conducted protein phosphokinase screening to analyze signaling pathway alterations induced by gastrodin in ataxin-3-77Q-expressing cells. A total of 37 kinases and 2 related total proteins were quantified, with 10 proteins showing differential expression between the gastrodin- and DMSO-treated groups (Table 1 , Fig. 4 ). These differentially expressed proteins were p38α (T180/Y182), eNOS (S1177), Src (Y419), lyn (Y397), GSK-3β (S9), ERK1/2 (T202/Y204, T185/Y187), HSP27 (S78/S82), Fgr (Y412), MSK1/2 (S376/S360), and PLC-γ1 (Y783). Further analysis focused on P38α, ERK1/2, their upstream kinases AKT1/2/3, and their downstream kinase p65, with their expression levels assessed using western blotting. Table 1 The list of proteome profile "hits" identified in the human phosphokinase array in the DMSO-treated and gastrodin-treated groups. Protein name Phosphorylation site Mean of differential value Rank 1 Mean of relative value to DMSO Rank 2 p38α T180/Y182 88345 1 3.8 16 eNOS S1177 85737.25 2 7.4 11 Src Y419 85212.5 3 23.9 2 lyn Y397 85115 4 21.5 3 GSK-3b S9 84765 5 -1.8 32 ERK1/2 T202/Y204,T185/Y187 84740 6 10.0 6 HSP27 S78/S82 84144.25 7 7.2 12 Fgr Y412 84077.5 8 8.6 9 MSK1/2 S376/S360 83952.75 9 12.1 4 PLC-γ1 Y783 83803.25 10 9.0 7 JNK1/2/3 T183/Y185,T221/Y223 83295 11 67.6 1 Yes Y426 83282.5 12 10.1 5 STAT2 Y689 82982.5 13 7.8 10 Lck Y394 82417.5 14 5.7 14 EGFR Y1086 82208 15 5.6 15 PDGF Rβ Y751 81863.25 16 6.6 13 CREB S133 80317.5 17 -10.7 34 STAT5a/b Y694/Y699 76605 18 -2.6 33 WNK1 T60 74275 19 -0.2 30 GSK-3a/b S21/S9 64150 20 -0.1 29 p53 S46 -46175 21 -0.8 31 p53 S15 -24700 22 8.9 8 p53 S392 -17275 23 -14.4 35 Akt1/2/3 T308 17152.5 24 1.5 20 PRAS40 T246 -14777.5 25 0.5 28 RSK1/2 S221/S227 10382.5 26 1.6 19 PYK2 Y402 6323.75 27 1.8 17 Chk-2 T68 6095 28 1.2 22 STAT3 Y727 -4802.5 29 0.8 27 p70 S6 Kinase T421/S424 4397.5 30 1.1 24 p70 S6 Kinase T389 2402.5 31 1.2 23 STAT1 Y701 2392.5 32 1.3 21 RSK1/2/3 S380/S386/S377 1748.75 33 1.1 26 Akt1/2/3 S473 1360 34 1.1 25 c-Jun S63 1262.5 35 1.7 18 Note: A total of 45 related proteins were included in the assay, but only 32 could be quantified based on the visible signal. The "Mean of differential value" is the average difference between the gastrodin-treated group and the DMSO-treated group. A lower value indicates a decrease in pathway signal. "Rank1" shows the ranking based on the differential values. The "Mean of the relative value to DMSO" is the average relative value of the gastrodin-treated group compared to the DMSO-treated group. "Rank2" shows the ranking based on the relative values to DMSO. "-" indicates no signal in the DMSO-treated group. The effect of gastrodin on cell models was tested. Gastrodin treatment significantly decreased phosphorylated or total ERK and total p38 protein levels (Fig. 5 D-F, Supplementary Fig. 1). Phosphorylated AKT1/2/3 levels did not change with gastrodin treatment (Fig. 5 B). Total levels of upstream AKT1/2/3 kinases showed an increasing trend with gastrodin treatment in HEK293T cells expressing ataxin-3-77Q (P = 0.18, Fig. 5 C). Total levels of p65, a downstream kinase, were significantly increased by gastrodin treatment in HEK293T cells expressing ataxin-3-15Q. However, p65 levels in HEK293T cells expressing ataxin-3-77Q increased after gastrodin treatment without significance (Fig. 5 G). These results suggest that the ERK1/2/P38 pathway, especially the total ERK1/2 protein level, was altered by gastrodin treatment in the SCA3 cell model expressing ataxin-3-77Q. Discussion Gastrodin, the main active ingredient in Gastrodia elata Blume, has been traditionally used to treat dizziness and epilepsy. It also shows potential in protecting against neurodegenerative diseases like Parkinson's and Alzheimer's. Previous research has demonstrated that gastrodin can inhibit aggregation and reduce toxicity in diseases like Alzheimer's and ALS [ 14 ]. However, there is currently no information on the relationship between gastrodin and SCA3. This study aims to investigate the impact of gastrodin on an SCA3 cell model. Key mechanistic findings and implications : Gastrodin is safe for use in SCA3 cell models. Its mechanism of action in treating SCA3 is believed to be related to its antioxidant properties. Gastrodin acts as an antioxidant, helping regulate the formation of aggregates in cells [ 15 – 16 ]. In our study, 10 µM of gastrodin increased its antioxidant activity in cells. Phosphokinase profiling revealed that gastrodin's neuroprotective effects in SCA3 cellular models involve modulation of multiple signaling nodes. 10 differentially regulated phosphoproteins were identified in ataxin-3-77Q-expressing cells after gastrodin treatment. Gastrodin may regulate phospho-kinases in the MAPK pathway (ERK1/2, p38α), downstream proteins (MSK1/2, HSP27), Src pathway (Src, Fgr, Lyn), and other pathways (eNOS pathway). The coordinated downregulation of the ERK1/2-p38-p65 axis is significant, suggesting it as a central pathway influenced by gastrodin. Therefore, gastrodin may play a role in SCA3 by modulating phospho-kinases. ERK1/2 Suppression as a Core Mechanism : The MAPK pathway regulates cellular processes such as inflammation, cell stress response, differentiation, and apoptosis. It is implicated in diseases like cancer, immune disorders, and neurodegenerative diseases. Reactive oxygen species can activate proteins in the MAPK pathway [ 17 ]. ERK, a member of the MAPK pathway, plays a role in cell proliferation, differentiation, and survival. Studies have shown that gastrodin can reduce pathological ERK overactivation by inhibiting ERK phosphorylation and regulating ERK protein stability. This dual mechanism of action is unique as most kinase modulators target activation states only. Previous research has demonstrated that ERK inhibition can reduce Tau aggregation in Alzheimer's disease and mitigate TDP-43 toxicity in ALS [ 18 ]. Gastrodin has been shown to decrease ERK/JNK MAPK expression and promote Nrf2 expression in cases of hepatotoxicity [ 19 ]. Additionally, gastrodin has been found to reduce neurotoxicity in diabetic mice by inhibiting the expression of p-ERK1/2, p-MEK1/2, BDNF, and TrkB [ 20 ]. These findings suggest that decreased ERK1/2 pathway activation may play a role in the protective effects of gastrodin in SCA3 cell models [ 21 ]. p38 Pathway Modulation : P38α, a member of the MAPK family, is involved in autophagy and apoptosis. It is activated by dual phosphorylation at Thr180 and Tyr182. Inflamed SCA3 cells show activation of NF-κB, JNK/JUN, and P38/STAT1 pathways, and inhibiting these pathways reduces aggregates in patients with AD or SCA3 [ 22 ]. A p38α MAPK inhibitor can clear protein aggregates by acting as a proteasome activator or autophagy inducer in an mTOR-dependent manner [ 23 ]. P38α inhibition can also clear aggregates via the Nrf2 pathway [ 24 ]. Clinical and preclinical evidence suggests p38α as a potential neurotherapeutic target [ 25 ]. Gastrodin may disrupt p38 signaling at the expression level, leading to a reduction in total p38 protein. This suppression could contribute to gastrodin's cytoprotective effects by potentially inhibiting stress-induced apoptosis in neurodegenerative contexts. Upstream/Downstream Signaling Context : ERK1/2 and eNOS function downstream of AKT [ 26 ]. The level of phosphorylated AKT was significantly lower in HEK293T cells overexpressing ataxin-3-15Q compared to control cells, and slightly lower in cells overexpressing ataxin-3-77Q (data not shown). Normal ataxin-3 appeared to increase total AKT protein level, while expanded ataxin-3 decreased it. This suggests that reduced AKT pathway activity may contribute to degeneration in SCA3. The non-significant increase in total AKT without changes in phosphorylation levels suggests AKT may not be the primary target of gastrodin, but could have ancillary roles. p65, known as RelA, is regulated by p38 [ 27 ]. The protein level of p65, a member of the NF-κB family that regulates inflammatory responses, was found to be lower in HEK293T cells overexpressing ataxin-3-77Q compared to wild-type cells (data not shown). Gastrodin increased p65 protein levels in HEK293T cells overexpressing ataxin-3-15Q but not in HEK293T cells or in HEK293T cells overexpressing ataxin-3-77Q. This suggests that polyQ expansion may impair NF-κB activation and warrants further investigation into gastrodin's role in inflammation regulation across disease states [ 28 ]. Pathway Integration and Disease Relevance : Our findings suggest that the coordinated suppression of ERK1/2 and p38 is in line with the known crosstalk between these MAPK pathways in mediating PolyQ-induced proteotoxicity, transcriptional dysregulation (via p65/NF-κB), and ER stress responses. Gastrodin may alleviate SCA3 pathogenesis by reducing hyperactivated MAPK signaling cascades, particularly ERK/p38 in mutant cells, indicating its potential as a disease-modifying agent. Limitations and Future Perspectives : The study has limitations including the use of HEK293T cells only, and validation in patient-derived neurons and in vivo models is necessary. Several questions remain unanswered: (1) The exact mechanism of ERK downregulation needs to be clarified, whether it is through proteasomal degradation or transcriptional regulation; (2) Further investigation is needed to understand how polyQ expansion disrupts p65 activation in response to gastrodin; (3) The potential synergistic effects of combining ERK/p38 inhibition with gastrodin treatment need to be systematically evaluated. Conclusion The study suggests that gastrodin can mitigate ataxin-3-77Q toxicity by modulating the ERK1/2/p38/p65 pathway. The decrease in total ERK1/2 protein indicates a new aspect of gastrodin's pharmacology, showing its potential as a regulator of pathogenic signaling in SCA3. These findings support the use of gastrodin in treating SCA3 and offer a new therapeutic approach for patients. Methods Cell culture and transfection Human embryonic kidney 293T (HEK293T) cells were generously gifted by Shaoan Xue from University College London and were maintained as previously described [ 29 ]. Three types of plasmids with the pEGFP-N2 vector, pEGFP-N2-MJD15Q, pEGFP-N2-MJD77Q, and pEGFP-N2-MJD148Q were gifted by Thorsten Schmidt from Tuebingen University [ 30 ]. Cells were transiently transfected with these plasmids using Lipo8000™ transfection reagent, as previously described [ 30 ]. Cells were exposed to gastrodin (CAS: 62499-27-8, Beijing Solarbio Science & Technology Co., Ltd.) or DMSO (CAS: 67-68-5, Beijing Solarbio Science & Technology Co., Ltd.) for different durations after 48 hours of transfection. Cell Counting Kit-8 (CCK-8) assay Cells overexpressing ataxin-3-77Q as a SCA3 cell model were used to perform CCK-8 assay as previously described [ 30 ]. Filter trap assay A filter trap assay was used to detect SDS-resistant insoluble mutant ataxin-3 aggregates as previously described [ 29 ]. Proteome Profiler Human Phospho-Kinase Array The Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems) is a membrane-based sandwich immunoassay. The array was divided into two parts (A and B) to maximize sensitivity and minimize cross-reactivity, detecting the relative levels of phosphorylation of 37 kinase phosphorylation sites and 2 related total proteins. At least 1 × 10 7 HEK293T cells overexpressing ataxin-3-77Q were cultured in the presence or absence of gastrodin for 24 hours. The cells were collected on ice and pelleted. Protein phosphorylation was analyzed using this kit according to the manufacturer's instructions. Signals were measured after adding an enhanced chemiluminescence (ECL) reagent (Affinity, KF8003) using Image Studio™ software on a LICOR instrument (LI-COR Biosciences, Lincoln, Nebraska, USA). All values, minus the average of the reference spot (loading control) values, were divided by two to obtain the final value. Then, the relative values were calculated as the final value in the DMSO-treated group minus the value in the gastrodin-treated group. SDS‒PAGE and western blot Protein expression was evaluated using western blotting. The process was reported in a previous study [ 30 ]. Statistical analysis All statistical analyses were carried out in GraphPad Prism 9 (La Jolla, CA). The data from multiple independent experiments are expressed as the means ± standard errors (SEMs). Statistical significance was analyzed by Student's t-test for two groups or one-way ANOVA and multiple comparisons for more than two groups. These data are normally distributed. Significance levels are described as follows: P < 0.05*; P < 0.01**; P < 0.001***, except where noted. Declarations Ethics declaration This study exclusively utilized commercially available human cell lines (HEK293T) and did not involve human participants, human tissues, or animal experimentation. As such, ethical approval from an institutional review board (IRB) or ethics committee was not required under international guidelines and institutional policies governing purely in vitro cell line research. Consent for publication Not applicable. Data availability statement We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. Competing interests The authors declare that they have no competing interests. Funding Declaration This research was funded by the National Natural Science Foundation of China (82202067, 32270530), the Key Research Program in Shaanxi Province of China (2024SF-YBXM-042), the Basic Research Program in Qinghai Province of China (2024-ZJ-752), and the Provincial College Student Innovation and Entrepreneurship Training Program (S202411080063, S202411080089). Author contributions Zijian Wang: Conceptualization, Funding acquisition, Project administration, Writing-original draft. Min Wang: Methodology. Ruitong Cheng: Methodology. Chan Wang: Methodology. Yingxun Liu: Data curation, Project administration.Fengqin He: Funding acquisition, Writing-review & editing. Xiaodong Xie: Writing-review & editing. Acknowledgements We would like to express our gratitude to Prof. Chen Jianjun from Lanzhou University for proofreading the manuscript. References Klockgether T, Mariotti C, Paulson HL. Spinocerebellar ataxia. Nat Rev Dis Primers. 5(1):24 (2019). Chen Z, et al. 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Trehalose prevents the formation of aggregates of mutant ataxin-3 and reduces soluble ataxin-3 protein levels in an SCA3 cell model. Neuroscience. 555:76-82 (2024). Supplementary Files SupplementaryFigure1.docx Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted Editorial decision: Minor revision 13 Aug, 2025 Reviewers agreed at journal 26 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor invited by journal 21 Jul, 2025 Editor assigned by journal 21 Jul, 2025 First submitted to journal 18 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7147978","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490361579,"identity":"83274279-ca75-4f4d-b515-a3e25b419900","order_by":0,"name":"Zijian Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYNACAzYeBgbmA0CWBRAnEK2FDaRUglgtYMBjQJwWg+NnD7/8UcAnwz+755vEzzYJBn72HAOGnzvwaDmTl2YhAXSYxJ2zmw17gVoke94YMPaewa3F7ECOmYEByC83cjc+4N0mwWBwI8eAmbENj5bzb8wMEoBa5G/kPDj4F6jFnqCWGznGDw4AtQANZ3wMtkWCgBb7G2/MGBuAWgxvpBkby/6T4JE486zgYC8eLZL9OcYff/w5Zi93I/mZ5JszNnL87ckbH/zEowUI2IBxcQzO4wERB/BqACaUDwwMNQTUjIJRMApGwYgGAK//TS5ThYZcAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3185-7432","institution":"Xi'an University","correspondingAuthor":true,"prefix":"","firstName":"Zijian","middleName":"","lastName":"Wang","suffix":""},{"id":490361580,"identity":"1c81c48c-d60b-4226-931f-a325c6c7b19f","order_by":1,"name":"Xunhao Xiao","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Xunhao","middleName":"","lastName":"Xiao","suffix":""},{"id":490361581,"identity":"817cd0db-e30a-429b-a0df-c3ad8e626e21","order_by":2,"name":"Min Wang","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Wang","suffix":""},{"id":490361582,"identity":"976e60c3-be9f-4623-910f-125d24053a9c","order_by":3,"name":"Ruitong Cheng","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Ruitong","middleName":"","lastName":"Cheng","suffix":""},{"id":490361583,"identity":"6b90a496-67f2-4e44-8cff-1d76d6df2212","order_by":4,"name":"Chan Wang","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Chan","middleName":"","lastName":"Wang","suffix":""},{"id":490361584,"identity":"60e67042-117d-4458-be30-3a371652202b","order_by":5,"name":"Yingxun Liu","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Yingxun","middleName":"","lastName":"Liu","suffix":""},{"id":490361585,"identity":"bf6d5bc5-eee0-4fc7-ada3-82e34ea9c354","order_by":6,"name":"Fengqin He","email":"","orcid":"","institution":"Xi'an University","correspondingAuthor":false,"prefix":"","firstName":"Fengqin","middleName":"","lastName":"He","suffix":""},{"id":490361586,"identity":"5c2f5d45-019e-432d-80cd-1325dd82509c","order_by":7,"name":"Xiaodong Xie","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-07-17 10:24:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7147978/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7147978/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13023-025-04089-1","type":"published","date":"2026-02-19T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87761604,"identity":"266ae128-2d4a-4292-b95b-1a4806cc98ac","added_by":"auto","created_at":"2025-07-28 17:04:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100884,"visible":true,"origin":"","legend":"\u003cp\u003eshows the impact of gastrodin on cytotoxicity in SCA3 cell models. HEK293T cells overexpressing ataxin-3-77CAG were treated with different concentrations of gastrodin for 2, 4, 6, 24, or 48 hours. The x-axis displays the treatment groups, while the y-axis shows the OD values normalized to the DMSO-treated group. The data were collected in triplicate for three independent experiments (n=3). Error bars indicate the standard error of the mean. The effects of each gastrodin concentration on DMSO-treated cells were analyzed using one-way ANOVA.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/0e9215975b53e39e49565f80.jpg"},{"id":87762365,"identity":"b349e241-cb75-457c-8117-d602452f9585","added_by":"auto","created_at":"2025-07-28 17:12:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94201,"visible":true,"origin":"","legend":"\u003cp\u003eA. Treatment with 50 µM of gastrodin decreased the total amount of aggregated ataxin-3, as shown by filter trap assay results. Representative dot images of filter traps are displayed. Bar graphs indicate values relative to the DMSO-treated group (N=3). B. Cell fractionation was performed according to Koch et al. Gastrodin treatment tended to decrease the amount of formic acid (FA)-soluble aggregates (large inclusions) and increase the amount of Triton X-100 (soluble protein) and SDS soluble aggregates (small inclusions) of expanded ataxin-3. C-D. Gastrodin at concentrations of 10 µM or 50 µM was tested for its effect on soluble ataxin-3 protein levels after 4 hours (C) or 24 hours of treatment (D) . Western blot images are provide. Bar graphs illustrate the impact of gastrodin on normal ataxin-3 (15Q) and expanded ataxin-3 (77Q) (N=3). The x-axis in panel A and C-D shows different treatment groups. The y-axis in panel A represents ataxin-3 signal (1H9) normalized to the DMSO-treated group. The y-axis in panel C-D represents ataxin-3 signal (1H9) relative to the loading control tubulin normalized to the DMSO-treated group. One-way ANOVA and multiple comparisons were conducted.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/a5aa186ddb98d640e65abed7.jpg"},{"id":87762554,"identity":"971179f5-901b-41ed-a734-55288f7e37f7","added_by":"auto","created_at":"2025-07-28 17:20:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43283,"visible":true,"origin":"","legend":"\u003cp\u003eshows the impact of different concentrations of gastrodin on the antioxidative activity of HEK293T cells (A), HEK293T cells overexpressing ataxin-3-15Q (B), and HEK293T cells overexpressing ataxin-3-77Q (C). The x-axis displays the various treatment groups, while the y-axis indicates the total antioxidative activity of the samples compared to the DMSO-treated group using a standard curve with Trolox for normalization. The data is based on more than three independent experiments (n≥3) with technical duplicates, analyzed using one-way ANOVA and multiple comparisons. The significance level was set at P \u0026lt; 0.01**.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/764a0674994b7653f3d30836.jpg"},{"id":87762366,"identity":"6e390b3a-bac1-43b5-897a-a36bf9c5b9dd","added_by":"auto","created_at":"2025-07-28 17:12:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40723,"visible":true,"origin":"","legend":"\u003cp\u003eshows the results of a human phospho-kinase array used to detect phosphorylated proteins in HEK293T cell lysates expressing ataxin-3-77Q. Part A of the array was incubated with 200 μg of cell lysate from HEK293T cells expressing ataxin-3-77Q that were either exposed to 50 µM gastrodin or to DMSO. Part B shows the top ten proteome profiler \"hits\" identified in the human phosphokinase array between the gastrodin-treated group and the DMSO-treated group. The data were obtained from two independent experiments.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/92b1a2416ee3cabf36f829ae.jpg"},{"id":87762367,"identity":"a2465238-63ca-443a-8e8b-748c51b2682d","added_by":"auto","created_at":"2025-07-28 17:12:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112001,"visible":true,"origin":"","legend":"\u003cp\u003eshows differences in protein levels among HEK293T cells, ataxin-3-15Q-expressing HEK293T cells, and ataxin-3-77Q-expressing HEK293T cells. Representative immunoblots and graphs demonstrate protein levels in the AKT, ERK, P38, and P65 pathways. Statistically significant differences are indicated by asterisks (*p \u0026lt; 0.05, ***p \u0026lt; 0.0001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/fcebf9eddecc44af14bf4e54.jpg"},{"id":103252283,"identity":"d54b72aa-07d7-49d2-8f6c-1d51304b6974","added_by":"auto","created_at":"2026-02-23 16:14:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1260043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/1a601a0d-e28d-4271-93de-532a416859f6.pdf"},{"id":87762370,"identity":"d7f897ba-e121-4f92-b20b-99d43f2f2b4d","added_by":"auto","created_at":"2025-07-28 17:12:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":195470,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7147978/v1/463e151f5a05064262a8b389.docx"}],"financialInterests":"","formattedTitle":"Gastrodin inhibits the formation of ataxin-3 aggregates by regulating the level of ERK1/2/P38 proteins","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. Gastrodin\u0026apos;s safety profile was demonstrated in SCA3 cellular models at concentrations up to 100\u0026nbsp;\u0026mu;M without causing cytotoxicity.\u003c/p\u003e\n\u003cp\u003e2. Gastrodin significantly reduced the formation of polyQ-expanded ataxin-3 aggregates.\u003c/p\u003e\n\u003cp\u003e3. There was a dose-dependent increase in soluble ataxin-3 levels by gastrodin.\u003c/p\u003e\n\u003cp\u003e4. Gastrodin was found to attenuate SCA3 proteotoxicity by simultaneously decreasing the total protein levels of ERK1/2 and p38.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSpinocerebellar ataxia type 3 (SCA3/Machado-Joseph disease), the most prevalent autosomal-dominant inherited ataxia globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], arises from abnormal polyglutamine (polyQ) expansion in ataxin-3. This mutation triggers cytotoxic protein aggregation-a hallmark of polyQ disorders - where misfolded ataxin-3 forms insoluble inclusions that sequester vital cellular components and drive neurodegeneration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Critically, no disease-modifying therapies currently exist to halt SCA3 progression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePathogenic synergy between proteotoxicity and oxidative stress underpins SCA3 pathogenesis. PolyQ-expanded ataxin-3 directly impairs proteostasis by overwhelming autophagy and ubiquitin-proteasome systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Concurrently, it induces mitochondrial dysfunction and redox imbalance, evidenced by elevated oxidative stress markers and diminished antioxidant defenses in patients and models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This dual insult creates a vicious cycle: oxidative stress accelerates aggregate formation, while aggregates further amplify oxidative damage. Existing therapeutic strategies targeting aggregation (e.g., autophagy enhancers, proteasome activators) or oxidative stress show preclinical promise but face translational barriers including safety concerns and poor blood-brain barrier (BBB) penetration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Repurposing phytochemicals with established neuroprotective profiles offers a pragmatic alternative [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGastrodin, the primary bioactive compound from Gastrodia elata Blume, emerges as a compelling candidate due to its multimodal neuroprotection, including demonstrated antioxidant, anti-aggregation, and anti-apoptotic effects in Alzheimer's and Parkinson's models [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; its favorable pharmacokinetics, including proven blood-brain barrier permeability with minimal side effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; and its unmet potential, never before evaluated in polyQ disorders. Here, we bridge this gap by investigating gastrodin's efficacy against SCA3 proteotoxicity. Using HEK293T-based models expressing physiologic (15Q) or pathogenic (77Q) ataxin-3, we assessed its effects on cell viability, aggregate burden, and soluble ataxin-3 levels, mapped kinase signaling alterations via phosphoproteomic screening, mechanistically dissected its regulation of the ERK1/2-p38 axis - a convergence point for oxidative stress and proteostasis disruption.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGastrodin at concentrations ranging from 5 \u0026micro;M to 100 \u0026micro;M is safe in HEK293T cells overexpressing ataxin-3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effect of gastrodin on cellular toxicity in SCA3 cell models, two methods, the CCK-8 method and the MTT assay, were used to assess cell proliferation and cell toxicity. CCK-8 assays revealed that gastrodin at concentrations of 5-100 \u0026micro;M did not have cytotoxic effects on cells after 2, 4, 6, 24, or 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E). The MTT results were consistent with the CCK-8 assay results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings indicate that gastrodin is considered safe for use in these SCA3 cell models, with concentrations ranging from 5-100 \u0026micro;M.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGastrodin reduces polyQ-expanded ataxin-3 aggregates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the effect of gastrodin on attenuating the aggregates formed by expanded ataxin-3 in a cell model of SCA3, a filter trap assay was employed, and a cell fractionation analysis was performed. Based on the cytotoxicity results and reports in the literature, we selected a gastrodin concentration of 10 \u0026micro;M or 50 \u0026micro;M, which has a protective effect on other neurodegenerative diseases. Filter trap assessment revealed that, compared with DMSO treatment, gastrodin treatment significantly decreased the number of aggregates formed by expanded ataxin-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTo analyze the population of aggregates decreased by gastrodin, we fractionated the aggregated ataxin-3 using a fractionation assay. Consistently, treatment with gastrodin decreased the amount of insoluble expanded ataxin-3 aggregates observed in formic acid (FA)-treated cells, which is an SDS-insoluble protein that is harmful to cells. Moreover, after gastrodin treatment, the levels of the Triton X-100-soluble and SDS-soluble ataxin-3 proteins increased, indicating that the degraded aggregates formed soluble proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFurther analyses were conducted to investigate the effect of gastrodin on the total level of soluble ataxin-3 protein. Western blot analysis revealed that 10 or 50 \u0026micro;M gastrodin treatment for 4 or 24 hours significantly increased soluble expanded ataxin-3 levels, while exerting minimal effects on normal ataxin-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC.D). These findings suggest that gastrodin effectively prevents aggregate formation in expanded ataxin-3-expressing HEK293T cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGastrodin plays a protective role through the ERK1/2/P38 signaling pathway.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the mechanism underlying the inhibitory effect of gastrodin on aggregates, we first used the total antioxidant capacity assay to assess the antioxidative ability of gastrodin in three cells models. Consistent with previous reports, compared with DMSO treatment, 10 \u0026micro;M gastrodin treatment increased the antioxidative activity in HEK293T cells, 10 \u0026micro;M and 50 \u0026micro;M gastrodin treatment showed an increased the tendency of the antioxidative activity in ataxin-3-15Q-expressing HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). However, gastrodin did not exhibit antioxidative activity in ataxin-3-77Q-expressing HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data demonstrated that gastrodin has the antioxidative activity, however, may also play a protective role in other signaling pathways in SCA3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe conducted protein phosphokinase screening to analyze signaling pathway alterations induced by gastrodin in ataxin-3-77Q-expressing cells. A total of 37 kinases and 2 related total proteins were quantified, with 10 proteins showing differential expression between the gastrodin- and DMSO-treated groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These differentially expressed proteins were p38α (T180/Y182), eNOS (S1177), Src (Y419), lyn (Y397), GSK-3β (S9), ERK1/2 (T202/Y204, T185/Y187), HSP27 (S78/S82), Fgr (Y412), MSK1/2 (S376/S360), and PLC-γ1 (Y783). Further analysis focused on P38α, ERK1/2, their upstream kinases AKT1/2/3, and their downstream kinase p65, with their expression levels assessed using western blotting.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe list of proteome profile \"hits\" identified in the human phosphokinase array in the DMSO-treated and gastrodin-treated groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhosphorylation site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean of differential value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRank\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean of relative value to DMSO\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRank\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep38α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT180/Y182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e88345\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeNOS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS1177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85737.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSrc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY419\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85212.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elyn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY397\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGSK-3b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84765\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eERK1/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT202/Y204,T185/Y187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84740\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHSP27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS78/S82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84144.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFgr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY412\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84077.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMSK1/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS376/S360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83952.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePLC-γ1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY783\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83803.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJNK1/2/3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT183/Y185,T221/Y223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e67.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83282.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSTAT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY689\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82982.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLck\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY394\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82417.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEGFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY1086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82208\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDGF Rβ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY751\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81863.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCREB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80317.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-10.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSTAT5a/b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY694/Y699\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76605\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWNK1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e74275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGSK-3a/b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS21/S9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-46175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-24700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS392\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-17275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-14.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAkt1/2/3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17152.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePRAS40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-14777.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRSK1/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS221/S227\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10382.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePYK2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY402\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6323.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChk-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6095\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSTAT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY727\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-4802.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep70 S6 Kinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT421/S424\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4397.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep70 S6 Kinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT389\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2402.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSTAT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY701\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2392.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRSK1/2/3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS380/S386/S377\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1748.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAkt1/2/3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS473\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec-Jun\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1262.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote:\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA total of 45 related proteins were included in the assay, but only 32 could be quantified based on the visible signal.\u003c/p\u003e\u003cp\u003eThe \"Mean of differential value\" is the average difference between the gastrodin-treated group and the DMSO-treated group. A lower value indicates a decrease in pathway signal.\u003c/p\u003e\u003cp\u003e\"Rank1\" shows the ranking based on the differential values.\u003c/p\u003e\u003cp\u003eThe \"Mean of the relative value to DMSO\" is the average relative value of the gastrodin-treated group compared to the DMSO-treated group.\u003c/p\u003e\u003cp\u003e\"Rank2\" shows the ranking based on the relative values to DMSO.\u003c/p\u003e\u003cp\u003e\"-\" indicates no signal in the DMSO-treated group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effect of gastrodin on cell models was tested. Gastrodin treatment significantly decreased phosphorylated or total ERK and total p38 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F, Supplementary Fig.\u0026nbsp;1). Phosphorylated AKT1/2/3 levels did not change with gastrodin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Total levels of upstream AKT1/2/3 kinases showed an increasing trend with gastrodin treatment in HEK293T cells expressing ataxin-3-77Q (P\u0026thinsp;=\u0026thinsp;0.18, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Total levels of p65, a downstream kinase, were significantly increased by gastrodin treatment in HEK293T cells expressing ataxin-3-15Q. However, p65 levels in HEK293T cells expressing ataxin-3-77Q increased after gastrodin treatment without significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These results suggest that the ERK1/2/P38 pathway, especially the total ERK1/2 protein level, was altered by gastrodin treatment in the SCA3 cell model expressing ataxin-3-77Q.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastrodin, the main active ingredient in Gastrodia elata Blume, has been traditionally used to treat dizziness and epilepsy. It also shows potential in protecting against neurodegenerative diseases like Parkinson's and Alzheimer's. Previous research has demonstrated that gastrodin can inhibit aggregation and reduce toxicity in diseases like Alzheimer's and ALS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, there is currently no information on the relationship between gastrodin and SCA3. This study aims to investigate the impact of gastrodin on an SCA3 cell model.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKey mechanistic findings and implications\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eGastrodin is safe for use in SCA3 cell models. Its mechanism of action in treating SCA3 is believed to be related to its antioxidant properties. Gastrodin acts as an antioxidant, helping regulate the formation of aggregates in cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In our study, 10 \u0026micro;M of gastrodin increased its antioxidant activity in cells. Phosphokinase profiling revealed that gastrodin's neuroprotective effects in SCA3 cellular models involve modulation of multiple signaling nodes. 10 differentially regulated phosphoproteins were identified in ataxin-3-77Q-expressing cells after gastrodin treatment. Gastrodin may regulate phospho-kinases in the MAPK pathway (ERK1/2, p38α), downstream proteins (MSK1/2, HSP27), Src pathway (Src, Fgr, Lyn), and other pathways (eNOS pathway). The coordinated downregulation of the ERK1/2-p38-p65 axis is significant, suggesting it as a central pathway influenced by gastrodin. Therefore, gastrodin may play a role in SCA3 by modulating phospho-kinases.\u003c/p\u003e\u003cp\u003e\u003cb\u003eERK1/2 Suppression as a Core Mechanism\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe MAPK pathway regulates cellular processes such as inflammation, cell stress response, differentiation, and apoptosis. It is implicated in diseases like cancer, immune disorders, and neurodegenerative diseases. Reactive oxygen species can activate proteins in the MAPK pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. ERK, a member of the MAPK pathway, plays a role in cell proliferation, differentiation, and survival. Studies have shown that gastrodin can reduce pathological ERK overactivation by inhibiting ERK phosphorylation and regulating ERK protein stability. This dual mechanism of action is unique as most kinase modulators target activation states only. Previous research has demonstrated that ERK inhibition can reduce Tau aggregation in Alzheimer's disease and mitigate TDP-43 toxicity in ALS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Gastrodin has been shown to decrease ERK/JNK MAPK expression and promote Nrf2 expression in cases of hepatotoxicity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, gastrodin has been found to reduce neurotoxicity in diabetic mice by inhibiting the expression of p-ERK1/2, p-MEK1/2, BDNF, and TrkB [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These findings suggest that decreased ERK1/2 pathway activation may play a role in the protective effects of gastrodin in SCA3 cell models [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ep38 Pathway Modulation\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eP38α, a member of the MAPK family, is involved in autophagy and apoptosis. It is activated by dual phosphorylation at Thr180 and Tyr182. Inflamed SCA3 cells show activation of NF-κB, JNK/JUN, and P38/STAT1 pathways, and inhibiting these pathways reduces aggregates in patients with AD or SCA3 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A p38α MAPK inhibitor can clear protein aggregates by acting as a proteasome activator or autophagy inducer in an mTOR-dependent manner [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. P38α inhibition can also clear aggregates via the Nrf2 pathway [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Clinical and preclinical evidence suggests p38α as a potential neurotherapeutic target [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Gastrodin may disrupt p38 signaling at the expression level, leading to a reduction in total p38 protein. This suppression could contribute to gastrodin's cytoprotective effects by potentially inhibiting stress-induced apoptosis in neurodegenerative contexts.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUpstream/Downstream Signaling Context\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eERK1/2 and eNOS function downstream of AKT [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The level of phosphorylated AKT was significantly lower in HEK293T cells overexpressing ataxin-3-15Q compared to control cells, and slightly lower in cells overexpressing ataxin-3-77Q (data not shown). Normal ataxin-3 appeared to increase total AKT protein level, while expanded ataxin-3 decreased it. This suggests that reduced AKT pathway activity may contribute to degeneration in SCA3. The non-significant increase in total AKT without changes in phosphorylation levels suggests AKT may not be the primary target of gastrodin, but could have ancillary roles.\u003c/p\u003e\u003cp\u003ep65, known as RelA, is regulated by p38 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The protein level of p65, a member of the NF-κB family that regulates inflammatory responses, was found to be lower in HEK293T cells overexpressing ataxin-3-77Q compared to wild-type cells (data not shown). Gastrodin increased p65 protein levels in HEK293T cells overexpressing ataxin-3-15Q but not in HEK293T cells or in HEK293T cells overexpressing ataxin-3-77Q. This suggests that polyQ expansion may impair NF-κB activation and warrants further investigation into gastrodin's role in inflammation regulation across disease states [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathway Integration and Disease Relevance\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eOur findings suggest that the coordinated suppression of ERK1/2 and p38 is in line with the known crosstalk between these MAPK pathways in mediating PolyQ-induced proteotoxicity, transcriptional dysregulation (via p65/NF-κB), and ER stress responses. Gastrodin may alleviate SCA3 pathogenesis by reducing hyperactivated MAPK signaling cascades, particularly ERK/p38 in mutant cells, indicating its potential as a disease-modifying agent.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations and Future Perspectives\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe study has limitations including the use of HEK293T cells only, and validation in patient-derived neurons and in vivo models is necessary. Several questions remain unanswered: (1) The exact mechanism of ERK downregulation needs to be clarified, whether it is through proteasomal degradation or transcriptional regulation; (2) Further investigation is needed to understand how polyQ expansion disrupts p65 activation in response to gastrodin; (3) The potential synergistic effects of combining ERK/p38 inhibition with gastrodin treatment need to be systematically evaluated.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study suggests that gastrodin can mitigate ataxin-3-77Q toxicity by modulating the ERK1/2/p38/p65 pathway. The decrease in total ERK1/2 protein indicates a new aspect of gastrodin's pharmacology, showing its potential as a regulator of pathogenic signaling in SCA3. These findings support the use of gastrodin in treating SCA3 and offer a new therapeutic approach for patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCell culture and transfection\u003c/p\u003e\u003cp\u003eHuman embryonic kidney 293T (HEK293T) cells were generously gifted by Shaoan Xue from University College London and were maintained as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Three types of plasmids with the pEGFP-N2 vector, pEGFP-N2-MJD15Q, pEGFP-N2-MJD77Q, and pEGFP-N2-MJD148Q were gifted by Thorsten Schmidt from Tuebingen University [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cells were transiently transfected with these plasmids using Lipo8000™ transfection reagent, as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cells were exposed to gastrodin (CAS: 62499-27-8, Beijing Solarbio Science \u0026amp; Technology Co., Ltd.) or DMSO (CAS: 67-68-5, Beijing Solarbio Science \u0026amp; Technology Co., Ltd.) for different durations after 48 hours of transfection.\u003c/p\u003e\u003cp\u003eCell Counting Kit-8 (CCK-8) assay\u003c/p\u003e\u003cp\u003eCells overexpressing ataxin-3-77Q as a SCA3 cell model were used to perform CCK-8 assay as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFilter trap assay\u003c/p\u003e\u003cp\u003eA filter trap assay was used to detect SDS-resistant insoluble mutant ataxin-3 aggregates as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProteome Profiler Human Phospho-Kinase Array\u003c/p\u003e\u003cp\u003eThe Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R\u0026amp;D Systems) is a membrane-based sandwich immunoassay. The array was divided into two parts (A and B) to maximize sensitivity and minimize cross-reactivity, detecting the relative levels of phosphorylation of 37 kinase phosphorylation sites and 2 related total proteins. At least 1 × 10\u003csup\u003e7\u003c/sup\u003e HEK293T cells overexpressing ataxin-3-77Q were cultured in the presence or absence of gastrodin for 24 hours. The cells were collected on ice and pelleted. Protein phosphorylation was analyzed using this kit according to the manufacturer's instructions. Signals were measured after adding an enhanced chemiluminescence (ECL) reagent (Affinity, KF8003) using Image Studio™ software on a LICOR instrument (LI-COR Biosciences, Lincoln, Nebraska, USA). All values, minus the average of the reference spot (loading control) values, were divided by two to obtain the final value. Then, the relative values were calculated as the final value in the DMSO-treated group minus the value in the gastrodin-treated group.\u003c/p\u003e\u003cp\u003eSDS‒PAGE and western blot\u003c/p\u003e\u003cp\u003eProtein expression was evaluated using western blotting. The process was reported in a previous study [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were carried out in GraphPad Prism 9 (La Jolla, CA). The data from multiple independent experiments are expressed as the means ± standard errors (SEMs). Statistical significance was analyzed by Student's t-test for two groups or one-way ANOVA and multiple comparisons for more than two groups. These data are normally distributed. Significance levels are described as follows: P \u0026lt; 0.05*; P \u0026lt; 0.01**; P \u0026lt; 0.001***, except where noted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study exclusively utilized commercially available human cell lines (HEK293T) and did not involve human participants, human tissues, or animal experimentation. As such,\u0026nbsp;ethical approval from an institutional review board (IRB) or ethics committee was not required\u0026nbsp;under international guidelines and institutional policies governing purely in vitro cell line research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (82202067, 32270530), the Key Research Program in Shaanxi Province of China (2024SF-YBXM-042), the Basic Research Program in Qinghai Province of China (2024-ZJ-752), and the Provincial College Student Innovation and Entrepreneurship Training Program (S202411080063, S202411080089).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZijian Wang: Conceptualization, Funding acquisition, Project administration, Writing-original draft. Min Wang: Methodology. Ruitong Cheng: Methodology. Chan Wang: Methodology. Yingxun Liu: Data curation, Project administration.Fengqin He: Funding acquisition, Writing-review \u0026amp; editing. Xiaodong Xie: Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Prof. Chen Jianjun from Lanzhou University for proofreading the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKlockgether T, Mariotti C, Paulson HL. Spinocerebellar ataxia. Nat Rev Dis Primers. 5(1):24 (2019).\u003c/li\u003e\n\u003cli\u003eChen Z, et al. Updated frequency analysis of spinocerebellar ataxia in China. Brain. 141(4): e22 (2018).\u003c/li\u003e\n\u003cli\u003eWarrick JM, et al. Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell. 93(6):939-49 (1998).\u003c/li\u003e\n\u003cli\u003eSchmidt T, et al. An isoform of ataxin-3 accumulates in the nucleus of neuronal cells in affected brain regions of SCA3 patients. Brain Pathol. 8(4):669-79 (1998).\u003c/li\u003e\n\u003cli\u003eMcLoughlin HS, et al. Antisense Oligonucleotide Silencing Reverses Abnormal Neurochemistry in Spinocerebellar Ataxia 3 Mice. Ann Neurol. 94(4):658-671 (2023).\u003c/li\u003e\n\u003cli\u003eReina CP, Zhong X, Pittman RN. Proteotoxic stress increases nuclear localization of ataxin-3. Hum Mol Genet. 19(2):235-49 (2010).\u003c/li\u003e\n\u003cli\u003ede Assis AM, et al. Peripheral Oxidative Stress Biomarkers in Spinocerebellar Ataxia Type 3/Machado-Joseph Disease. Front Neurol. 8:485 (2017).\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;zquez-Mojena Y, Le\u0026oacute;n-Arcia K, Gonz\u0026aacute;lez-Zaldivar Y, Rodr\u0026iacute;guez-Labrada R, Vel\u0026aacute;zquez-P\u0026eacute;rez L. Gene Therapy for Polyglutamine Spinocerebellar Ataxias: Advances, Challenges, and Perspectives. Mov Disord. 36(12):2731-2744 (2021).\u003c/li\u003e\n\u003cli\u003eDur\u0026atilde;es F, Pinto M, Sousa E. Old Drugs as New Treatments for Neurodegenerative Diseases. Pharmaceuticals (Basel). 11(2):44 (2018).\u003c/li\u003e\n\u003cli\u003eWang Y, et al. Focused Ultrasound Promotes the Delivery of Gastrodin and Enhances the Protective Effect on Dopaminergic Neurons in a Mouse Model of Parkinson\u0026apos;s Disease. Front Cell Neurosci. 16:884788 (2022).\u003c/li\u003e\n\u003cli\u003eZhang J, et al. Gastrodin programs an Arg-1+ microglial phenotype in hippocampus to ameliorate depression- and anxiety-like behaviors via the Nrf2 pathway in mice. Phytomedicine. 113:154725 (2023).\u003c/li\u003e\n\u003cli\u003eLin YE, et al. Glial Nrf2 signaling mediates the neuroprotection exerted by Gastrodia elata Blume in Lrrk2-G2019S Parkinson\u0026apos;s disease. Elife. 10:e73753 (2021).\u003c/li\u003e\n\u003cli\u003eLiu Y, et al. A Review on Central Nervous System Effects of Gastrodin. Front Pharmacol. 9:24 (2018).\u003c/li\u003e\n\u003cli\u003eXiao G, Tang R, Yang N, Chen Y. Review on pharmacological effects of gastrodin. Arch Pharm Res. 46(9-10):744-770 (2023).\u003c/li\u003e\n\u003cli\u003eHao S, et al. Glycosides and Their Corresponding Small Molecules Inhibit Aggregation and Alleviate Cytotoxicity of A\u0026beta;40. ACS Chem Neurosci. 13(6):766-775 (2022).\u003c/li\u003e\n\u003cli\u003eHuang Q, et al. Gastrodin: an ancient Chinese herbal medicine as a source for anti-osteoporosis agents via reducing reactive oxygen species. Bone. 73:132-44 (2015).\u003c/li\u003e\n\u003cli\u003eVarela L, Garcia-Rendueles MER. Oncogenic Pathways in Neurodegenerative Diseases. Int J Mol Sci. 23(6):3223 (2022).\u003c/li\u003e\n\u003cli\u003eYue W, et al. Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS. Immun Ageing. 20(1):27 (2023).\u003c/li\u003e\n\u003cli\u003eWang XL, et al. Gastrodin prevents motor deficits and oxidative stress in the MPTP mouse model of Parkinson\u0026apos;s disease: involvement of ERK1/2-Nrf2 signaling pathway. Life Sci. 114(2):77-85 (2014).\u003c/li\u003e\n\u003cli\u003eQi YH, et al. Early intervention with gastrodin reduces striatal neurotoxicity in adult rats with experimentally-induced diabetes mellitus. Mol Med Rep. 19(4):3114-3122 (2019).\u003c/li\u003e\n\u003cli\u003eSowa AS, et al. Neurodegenerative phosphoprotein signaling landscape in models of SCA3. Mol Brain. 14(1):57 (2021).\u003c/li\u003e\n\u003cli\u003eChiu YJ, et al. Pathomechanism characterization and potential therapeutics identification for SCA3 targeting neuroinflammation. 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Trends Biochem Sci. 36(6):320-8 (2011). \u003c/li\u003e\n\u003cli\u003eZhang D, et al. The Agpat4/LPA axis in colorectal cancer cells regulates antitumor responses via p38/p65 signaling in macrophages. Signal Transduct Target Ther. 5(1):24 (2020).\u003c/li\u003e\n\u003cli\u003eXing Y, Li L. Gastrodin protects rat cardiomyocytes H9c2 from hypoxia-induced injury by up-regulation of microRNA-21. Int J Biochem Cell Biol. 109:8-16 (2019).\u003c/li\u003e\n\u003cli\u003eWang ZJ, et al. Divalproex sodium modulates nuclear localization of ataxin-3 and prevents cellular toxicity caused by expanded ataxin-3. CNS Neurosci Ther. 24(5):404-411 (2018).\u003c/li\u003e\n\u003cli\u003eWang Z, et al. Trehalose prevents the formation of aggregates of mutant ataxin-3 and reduces soluble ataxin-3 protein levels in an SCA3 cell model. Neuroscience. 555:76-82 (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gastrodin, SCA3, Ataxin-3 aggregation, ERK/p38 signaling, Neuroprotection","lastPublishedDoi":"10.21203/rs.3.rs-7147978/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7147978/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eSpinocerebellar ataxia type 3 (SCA3/Machado-Joseph disease), an incurable autosomal dominant neurodegenerative disorder, is caused by cytotoxic aggregation of polyglutamine-expanded ataxin-3 protein. Novel therapeutic strategies targeting its pathogenesis are urgently needed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eGiven gastrodin's established antioxidative and neuroprotective properties, this study investigated its therapeutic potential against SCA3 pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eThree distinct cell models including parental HEK293T, ataxin-3-15Q (physiologic), and ataxin-3-77Q (pathogenic) were employed to assess gastrodin cytotoxicity, quantify insoluble aggregate formation and measure soluble ataxin-3 levels. Mechanistic studies included antioxidant capacity assays, human phosphokinase array profiling (37 kinases) and western blot validation of MAPK pathway components.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Gastrodin treatment showed no cytotoxicity, significantly suppressed ataxin-3-77Q aggregate accumulation (p\u0026lt;0.01), increased soluble ataxin-3 levels, enhanced cellular antioxidant capacity and selectively downregulated ERK1/2 and p38 proteins in MAPK pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWe provide first evidence that gastrodin mitigates polyQ-mediated proteotoxicity by reducing ataxin-3 aggregation through suppression of the ERK1/2-p38 signaling axis, revealing a novel mechanistic basis for SCA3 therapeutic development.\u003c/p\u003e","manuscriptTitle":"Gastrodin inhibits the formation of ataxin-3 aggregates by regulating the level of ERK1/2/P38 proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 17:04:03","doi":"10.21203/rs.3.rs-7147978/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2025-08-13T08:25:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-26T09:25:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T13:38:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Orphanet Journal of Rare Diseases","date":"2025-07-21T07:54:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T05:18:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Orphanet Journal of Rare Diseases","date":"2025-07-18T06:37:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39f95b98-e2e9-417d-bff0-9a93609ff1e8","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:10:27+00:00","versionOfRecord":{"articleIdentity":"rs-7147978","link":"https://doi.org/10.1186/s13023-025-04089-1","journal":{"identity":"orphanet-journal-of-rare-diseases","isVorOnly":false,"title":"Orphanet Journal of Rare Diseases"},"publishedOn":"2026-02-19 15:56:57","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-07-28 17:04:03","video":"","vorDoi":"10.1186/s13023-025-04089-1","vorDoiUrl":"https://doi.org/10.1186/s13023-025-04089-1","workflowStages":[]},"version":"v1","identity":"rs-7147978","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7147978","identity":"rs-7147978","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}