Inhibition of TGF-beta signaling protects from alpha-synuclein induced toxicity

Inhibition of TGF-beta signaling protects from alpha-synuclein induced toxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inhibition of TGF-beta signaling protects from alpha-synuclein induced toxicity Matthias Höllerhage, Oscar Wing Ho Chua, Linghan Duan, Svenja Bothe, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6867964/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract Parkinson’s disease (PD) is histopathologically defined by the presence of Lewy bodies, which are intracellular proteinaceous inclusions that contain mainly aggregated alpha-synuclein (aSyn). It is believed that oligomeric intermediates between monomeric aSyn and large aggregates are neurotoxic, which would lead to the demise of dopaminergic neurons. Therefore, novel therapies preventing aSyn-induced cell death need to be developed. Therefore, we performed a genome-wide siRNA screening in an aSyn-induced dopaminergic cell death model and found the knockdown of three Transforming Growth Factor-beta (TGFb) pathway-related genes to be protective. Hence, we hypothesized that a reduction in TGFb signaling would protect dopaminergic neurons from aSyn-induced toxicity. Thus, we validated the results of the genome-wide knockdown screening with the use of two different types of siRNAs. We confirmed that the knockdown of Activin receptor-like kinase 5 ( ALK5 ) and Mothers against decapentaplegic homolog 2 ( SMAD2 ), two genes of the TGFb pathway, protected dopaminergic neurons from aSyn-induced toxicity. An increase in TGFb signaling by treatment with TGFb ligands further exacerbated aSyn-induced toxicity, whereas this effect was mitigated by knockdown of ALK5 , SMAD2 , or Dynein light chain roadblock type-1 ( DYNLRB1 ). Moreover, TGFb ligand treatment induced an up-regulation of SNCA mRNA expression in aSyn-overexpressing cells. Interestingly, consistent with the literature, we identified an up-regulation of the genes of the TGFb pathway in aSyn-overexpressing cells. Altogether, we identified a potential protective role of the TGFb pathway against aSyn-induced toxicity. These findings provide a rationale for the development of novel strategies against PD. Health sciences/Diseases/Neurological disorders/Parkinson's disease Biological sciences/Molecular biology/RNAi Biological sciences/Cell biology/Cell signalling/Growth factor signalling Parkinson’s disease alpha-synuclein genome-wide RNAi screening Transforming Growth Factor-beta signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder. In PD, the death of dopaminergic neurons in the substantia nigra (SN) leads to a series of observable motor symptoms such as bradykinesia, resting tremor, and rigidity [ 1 ]. Histopathologically, PD is defined by the presence of Lewy bodies (LBs) in the brains of PD patients[ 2 ]. LBs are intracellular proteinaceous inclusions that mainly contain abnormally aggregated alpha-synuclein (aSyn) [ 3 , 4 ]. During the aggregation process of aSyn, oligomeric intermediates between monomers and large insoluble aggregates are formed, and these intermediates are believed to be toxic [ 5 , 6 ]. We performed a genome-wide siRNA screening in dopaminergic Lund Mesencephalic (LUHMES) cells [ 7 , 8 ] with moderate overexpression of wild-type (WT) aSyn [ 9 ], in which the knockdown of twelve genes was identified to protect from degeneration [ 10 ]. Among these, three genes are related to the Transforming Growth Factor-beta (TGFb) canonical pathway, namely Activin A receptor type 1C ( ACVR1C , also known as Activin receptor-like kinase 7, ALK7 ), Dynein light chain roadblock type-1 ( DYNLRB1 ), and Glycogen synthase kinase 3 beta ( GSK3B ). The TGFb superfamily is one of the largest signaling pathway families in humans, comprising 33 ligands, 5 type-II receptors, and 7 type-I receptors. Pathways of the TGFb superfamily can be classified according to the ligands, as well as by the downstream cascades. The pathway that involves the phosphorylation of SMAD proteins is known as the canonical pathway [ 11 ] (Fig. 1 ). The binding of TGFb ligands (3 isoforms in humans) to the dimeric TGFb type-II receptor (TGFBR2) activates the TGFb canonical pathway. Upon ligand binding, TGFBR2 recruits and phosphorylates the dimeric TGFb type-I receptor (TGFBR1, also known as activin receptor-like kinase 5, ALK5) [ 11 – 13 ]. Together they form a heterotetrameric complex that is internalized into early endosomes, where the heterotetrameric receptor complex can phosphorylate Mothers against decapentaplegic homolog 2 (SMAD2) and/or SMAD3 proteins [ 12 , 14 , 15 ]. The activated SMAD2/3 complex is then coupled to SMAD4 for nuclear translocation, where the SMAD complex binds its target genes and drives their expression together with a number of different transcription factors [ 16 ]. ALK7 and DYNLRB1 are closely related to the TGFb pathway, as ALK7 is a type-I receptor that specifically transduces signals from the ligand Nodal [ 17 ], and DYNLRB1 is involved in the nuclear translocation of phospho-SMAD2 [ 18 ]. GSK3B has been implicated to be involved in the degradation of SMAD3 [ 19 ]. An illustration depicting the TGFb pathway can be found on Fig. 1 . In the central nervous system, TGFb ligands and receptors are expressed in most parts of the brain, and they are reported to be involved in multiple cellular processes. It has been shown that TGFb ligand treatment could promote the survival and differentiation of primary mesencephalic dopaminergic neurons [ 20 , 21 ]. Moreover, TGFb signaling has been implicated to play a role in the regulation of neuronal proliferation and apoptosis [ 22 , 23 ]. Due to its large distribution and involvement in multiple cellular processes, several studies looked into whether an increase in TGFb signaling could protect dopaminergic neurons from the toxicity induced by the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP + ), both in vivo and in vitro [ 24 – 26 ]. However, results from these studies remain contradictory, and the role of TGFb signaling in the context of MPP + -induced toxicity remains inconclusive. In a previous work, our group discovered that the knockdown of genes involved in the TGFb signaling pathway protected LUHMES cells against aSyn-induced toxicity [ 10 ]. Hence, in this project, we aimed to investigate the role of the TGFb pathway upon aSyn overexpression. Results Validation of the results of the genome-wide siRNA screening We validated if the knockdown of ALK7 , DYNLRB1 , and GSK3B could protect cells from aSyn-induced toxicity in our standard cell culture conditions. Utilizing esiRNAs, as employed in the genome-wide screening, we aimed to validate the screening results. In addition, we used siPOOLs siRNAs to further confirm and consolidate our findings. Both types of siRNAs are ‘pools’ of siRNAs that are produced by enzymatic cleavage of long double-stranded RNA, in which these siRNAs all target the same mRNA. The use of these two distinct siRNA pools helped enhance the reliability of our outcomes. To analyze the changes in aSyn-related toxicity, LDH data were normalized relative to the LDH levels measured in aSyn-overexpressing cells. Thus, data from LDH released from aSyn-overexpressing cells are presented as 100%. We first validated the findings of the genome-wide siRNA screening [ 10 ] utilizing esiRNAs targeting ALK7 , DYNLRB1 , and GSK3B , which were highlighted in the primary screening. Notably, treatment with esiRNAs against ALK7 , DYNLRB1 , and GSK3B significantly protected LUHMES cells from aSyn-induced cytotoxicity (Fig. 2 A). This reduction was evident in the diminished LDH release as compared to untreated aSyn-overexpressing cells (esiRNA against ALK7 : 68.3 ± 2.2%, p < 0.001; esiRNA against DYNLRB1 : 72.6 ± 2.6%, p < 0.001; esiRNA against GSK3B : 76.5 ± 2.5%, p < 0.001; each compared to untransfected aSyn-overexpressing cells; 100 ± 1.5%). By using the siPOOL siRNAs, we also confirmed that the transfection with siPOOL siRNAs against DYNLRB1 and GSK3B protected aSyn-overexpressing cells (Fig. 2 B), as quantified by the reduction in LDH released from aSyn-overexpressing cells (siPOOL siRNA against DYNLRB1 : 74.3 ± 3.8%, p < 0.001; siPOOL siRNA against GSK3B : 87.7 ± 2.9%, p < 0.01; each compared to untransfected aSyn-overexpressing cells; 100 ± 1.6%). However, in contrast to the esiRNA, the treatment with the siPOOL siRNA against ALK7 did not protect aSyn-overexpressing cells (Fig. 2 B). To determine whether the protection against aSyn-induced toxicity was the consequence of a specific knockdown of the respective target genes, or an off-target effect, we investigated the expression of GSK3B, DYNLRB1, and ALK7 in untransduced cells and aSyn-overexpressing cells after the transfection with the siRNAs. By performing WB analyses, we confirmed that the esiRNA against GSK3B led to a significant down-regulation of GSK3B protein in both untransduced cells and aSyn-overexpressing cells (Fig. 2 C; full-size Western blot in Supplementary File 2 Fig. A). Similarly, the siPOOL siRNA against GSK3B markedly reduced the expression of GSK3B protein in both untransduced cells and aSyn-overexpressing cells (Fig. 2 D; full-size Western blot in Supplementary File 2 Fig. B). To reinforce our hypothesis that reducing GSK3B levels protects LUHMES cells from aSyn-induced toxicity, we tested treated LUHMES cells with two different inhibitors of GSK3B, tideglusib [ 27 ] and GSK3B inhibitor-VIII (GSK8) [ 28 ]. Both tideglusib and GSK8 led to a significant reduction in LDH release in aSyn-overexpressing cells, demonstrating their protective efficacy. Tideglusib reduced aSyn-induced toxicity at concentrations of 10 µM (88.8 ± 1.8, p < 0.01) and 20 µM (72.9 ± 3.3%, p < 0.001; untreated aSyn-overexpressing cells: 100 ± 2.03%; Supplementary Fig. S1 A). Similarly, GSK8 treatment resulted in a significant reduction of LDH release in aSyn-overexpressing cells (100 ± 2.9%) at concentrations of 1 µM (84.4 ± 4.3%, p < 0.01) and 2 µM (57.4 ± 2.8%, p < 0.001; Supplementary Fig. S1 B). Additionally, aSyn overexpression significantly elevated GSK3B levels as compared to untransduced cells, highlighting the role of GSK3B in aSyn-induced toxicity in our cell model. Collectively, our results suggest that reducing GSK3B availability, either through the knockdown of GSK3B or by direct pharmacological inhibition, protects LUHMES cells from aSyn-induced toxicity. Due to the lack of commercially available WB antibodies for DYNLRB1, we assessed the knockdown efficacy of both the esiRNAs and the siPOOL siRNAs against DYNLRB1 by qPCR. The esiRNA against DYNLRB1 significantly reduced DYNLRB1 mRNA expression in both untransduced cells and aSyn-overexpressing cells (Fig. 2 E). We also identified a significant up-regulation of DYNLRB1 associated with aSyn overexpression. Similarly, the siPOOL siRNA targeting DYNLRB1 led to a significant reduction in DYNLRB1 mRNA expression in untransduced cells and aSyn-overexpressing cells (Fig. 2 F), indicating that both siRNAs against DYNLRB1 were effective. Since transfection with the siPOOL siRNA against ALK7 did not protect against aSyn-induced toxicity, we conducted a deeper analysis of ALK7 expression in LUHMES cells. In contrast to the gene expression of the other two type-I receptors of the TGFb pathway ( ALK4 and ALK5 ), as well as the housekeeping genes ( huUBQLN1 and POLR2A ), ALK7 mRNA was only detectable after 34.6 ± 0.2 cycles, indicating extremely low expression levels in LUHMES cells (Fig. 2 G). Subsequently, we examined whether the esiRNA and the siPOOL siRNA targeting ALK7 could reduce ALK7 expression. The esiRNA against ALK7 significantly reduced ALK7 expression only in aSyn-overexpressing cells (Fig. 3 A). As the treatment with the esiRNA, but not the siPOOL siRNA against ALK7 , conferred protection to LUHMES cells against aSyn-induced toxicity, we hypothesized that the protectiveness observed with the esiRNA against ALK7 might arise from off-target activities. Considering the close phylogenetic similarity between ALK7 and the TGFb pathway receptors ALK4 and ALK5 [ 29 – 31 ], we hypothesized that ALK4 and ALK5 might be potential off-targets of the ALK7 esiRNA. However, our analysis revealed that the esiRNA against ALK7 did not down-regulate ALK4 mRNA levels in either untransduced cells or aSyn-overexpressing cells, suggesting that ALK4 was not the off-target of the ALK7 esiRNA (Fig. 3 B). In contrast, the esiRNA against ALK7 significantly reduced ALK5 mRNA expression in aSyn-overexpressing cells (Fig. 3 C), indicating that ALK5 was indeed an off-target. The siPOOL siRNA against ALK7 also resulted in a reduction in ALK7 expression only in aSyn-overexpressing cells (Fig. 3 D). However, unlike the esiRNA, the siPOOL siRNA against ALK7 did not lead to significant changes in the expression of both ALK4 and ALK5 in either untransduced cells or aSyn-overexpressing cells (Fig. 3 E, F). Given that the protection against aSyn-induced toxicity was only observed after the transfection of esiRNA against ALK7 , for which we identified ALK5 as off-target, together with the fact that the expression of ALK7 was very low in LUHMES cells (Fig. 2 G), we proposed that ALK5 knockdown, rather than ALK7 knockdown, was responsible for the observed protective effect against aSyn-induced toxicity. ALK5 and its downstream pathway protected LUHMES cells from aSyn-induced toxicity To verify that the knockdown of ALK5 protected against aSyn-induced toxicity, we first validated if the siPOOL siRNA against ALK5 could effectively and specifically reduce ALK5 mRNA expression. The siPOOL siRNA against ALK5 significantly reduced ALK5 expression in both untransduced cells and aSyn-overexpressing cells (Fig. 3 I). We also confirmed that the siPOOL siRNA against ALK5 did not lead to changes in the mRNA expression of ALK4 or ALK7 in both untransduced cells and aSyn-overexpressing cells (Fig. 3 G, H). We then confirmed that the knockdown of ALK5 could protect LUHMES cells from aSyn-induced toxicity (Fig. 4 A). The knockdown of ALK5 protected LUHMES cells from aSyn-induced toxicity, as quantified by a reduction of LDH release (from 100 ± 2.0% as in untreated aSyn-overexpressing cells to 87.2 ± 4.0%, p < 0.01). The protective effect of ALK5 knockdown was specific against aSyn-induced toxicity, since the knockdown of ALK5 did not lead to changes in cell viability in either untransduced cells or GFP-expressing cells (Fig. 4 A). Next, we used an inhibitor of ALK5, SM16 [ 32 , 33 ], to further confirm if the inhibition of ALK5 protected against aSyn-induced toxicity (Fig. 4 B). As anticipated, SM16 treatment led to a significant dose-dependent protection against aSyn-induced toxicity. In particular, SM16 treatment led to a reduction of LDH release in aSyn-overexpressing cells from 100 ± 2.2% to 77.5 ± 4.2% at 10 µM ( p < 0.001), to 68.1 ± 4.8% at 20 µM ( p < 0.001), and to 55.6 ± 4.8% at 40 µM ( p < 0.001). Similar to the observation made after the knockdown of ALK5 , the protection by the treatment with SM16 was specific to aSyn-overexpressing cells (Fig. 4 C). We then explored if the knockdown of SMAD2 and/or SMAD3 , the downstream targets of ALK5, could confer protection against aSyn-induced toxicity. Interestingly, only the knockdown of SMAD2 , but not SMAD3 , demonstrated a protective effect. When compared to the LDH released from untreated aSyn-overexpressing cells, the knockdown of SMAD2 led to a reduction of LDH release from 100 ± 3.4% to 88.2 ± 4.6% ( p < 0.05). Furthermore, the protection after the knockdown of SMAD2 was only observed in aSyn-overexpressing cells, indicating an aSyn-dependent effect (Fig. 4 D). Due to the high sequence homology between SMAD2 and SMAD3 [ 34 , 35 ], we verified the specificities of siPOOL siRNAs for their respective targets. In both untransduced cells and aSyn-overexpressing cells, we found that the siPOOL siRNA against SMAD2 specifically reduced SMAD2 expression without affecting the expression of SMAD3 (Supplementary Fig. S1 C). In the same manner, the siPOOL siRNA against SMAD3 specifically reduced SMAD3 expression without affecting the expression of SMAD2 (Supplementary Fig. S1 D). Interestingly, the expression of SMAD2 was much higher than SMAD3 (approximately 10 threshold cycles higher, i.e. over two orders of magnitude) in LUHMES cells (Supplementary Fig. S1 E). Since the TGFb pathway is involved in the differentiation of dopaminergic neurons [ 24 , 36 ], we performed a control experiment to verify that the knockdown of ALK5 , SMAD2 , SMAD3 , or DYNLRB1 did not interfere with the differentiation of LUHMES cells. We confirmed that the mRNA expression of tyrosine hydroxylase ( TH ), a dopaminergic neuronal marker, was not significantly altered by the knockdown of ALK5 , SMAD2 , SMAD3 , or DYNLRB1 in untransduced cells, aSyn-overexpressing cells, and GFP-expressing cells (Supplementary Fig. S1 F). To further ensure that the knockdown of ALK5 , SMAD2 , SMAD3 , or DYNLRB1 did not impact on the transcriptional activities of the adenoviral vectors used in our experiments, we measured the expression of aSyn on both transcript and protein levels. The knockdown of ALK5 , SMAD2 , SMAD3 , or DYNLRB1 did not affect the expression of SNCA , the gene that encodes aSyn, nor the level of aSyn protein (Supplementary Fig. S1 G, H; full-size Western blot in Supplementary File 2 Fig. C) in untransduced cells, aSyn-overexpressing cells, or GFP-expressing cells. Concurrently, the knockdown of ALK5 , SMAD2 , SMAD3 , or DYNLRB1 did not interfere with the expression of GFP in GFP-expressing cells (Supplementary Fig. S1 I). These data emphasize that the protection against aSyn-induced toxicity resulting from the knockdown of ALK5 , SMAD2 , or DYNLRB1 was not caused by an interference with differentiation of LUHMES cells, or by a change in the transcriptional activities of the adenoviral vectors. In addition, as SMAD3 expression was low in LUHMES cells and its knockdown did not yield observable phenotypic changes such as protection against aSyn-induced toxicity (Fig. 4 D) or changes in the expression of aSyn (Supplementary Fig. S1 G, H), we excluded SMAD3 in the following experiments. We then performed immunocytochemistry to investigate whether the knockdown of ALK5 , SMAD2 , and DYNLRB1 also ameliorated loss of neuronal network density (Fig. 4 F; untransduced cells: Supplementary Fig. S2 A; GFP-expressing cells: Supplementary Fig. S2 B). We quantified the density of neuronal network by counting the number of quadruple points per cell (Fig. 4 E). Each quadruple point reflects a dendritic junction that contains or connects four distinct neurite branches, serving as an indicator of neuronal network complexity. In untransduced cells and GFP-expressing cells, the knockdown of ALK5 , SMAD2 , or DYNLRB1 did not significantly alter neuronal network density, as compared to respective control cells without siRNA transfection (Fig. 4 G). As expected, aSyn overexpression substantially decreased the density of the neuronal network, as quantified by a reduction in the number of quadruple points per cell in comparison to the untransduced cells. (untransduced cells: 199.7 ± 10.7; aSyn-overexpressing cells: 84.6 ± 8.5; p < 0.001). Such reduction of the neuronal network complexity in aSyn-overexpressing cells was counteracted by the knockdown of ALK5 and SMAD2 , as we observed a significant increase in the number of quadruple points per cell in comparison to the untransfected aSyn-overexpressing cells ( ALK5 knockdown: 154.7 ± 11.4, p < 0.001; SMAD2 knockdown: 150.5 ± 8.4, p = 0.002; Fig. 4 E). On the other hand, the knockdown of DYNLRB1 also led to an increase in the density of neuronal network of aSyn-overexpressing cells, however it did not reach statistical significance (126.5 ± 13.2; Fig. 4 F). To further support these findings, we also detected activated caspases 3/7, an apoptotic marker, using a fluorescent dye (CellEvent™; Supplementary Fig. S2 C, D). In aSyn-overexpressing cells, the knockdown of SMAD2 led to an obvious reduction in the expression of caspase 3/7 (from 100 ± 10.0% to 65.3 ± 5.73%). In summary, we demonstrated that reducing TGFb signaling, specifically via the knockdown of ALK5 , SMAD2 , and DYNLRB1 , protected LUHMES cells against aSyn-induced toxicity. In particular, the knockdown of ALK5 prevented the loss of neuronal network density, whereas the knockdown of SMAD2 not only preserved neuronal network density but also inhibited apoptotic activation. The over-activation of TGFb pathway is detrimental to aSyn-overexpressing cells Since the inhibition of TGFb signaling protected against aSyn-induced toxicity, we explored whether activating this pathway would have the opposite effect. Therefore, we treated aSyn-overexpressing cells with recombinant human TGFb proteins. We used TGFb ligands at 10 ng/mL, a dose that was previously reported to effectively activate the TGFb pathway [ 37 , 38 ]. In comparison to untreated aSyn-overexpressing cells (indicated as 100%), treatment with TGFb1, TGFb2, and TGFb3 led to an increase of LDH release in aSyn-overexpressing cells (TGFb1: 113.3 ± 2.0%, p < 0.05; TGFb2: 114.4 ± 2.2%, p < 0.05; TGFb3: 112.6 ± 2.5%, p = 0.01; Fig. 5 A-C). Furthermore, the increase in cytotoxicity induced by TGFb ligand treatment was also reflected by a decrease in neuronal network density. Specifically, following the treatment with TGFb1, we observed a significant decrease in the number of quadruple points per cell, dropping from 109.9 ± 4.5 in untreated aSyn-overexpressing cells, to 72.9 ± 7.2 ( p = 0.03; Fig. 5 D, E). Notably, this cytotoxic effect caused by TGFb ligand treatment was exclusive to aSyn-overexpressing cells. In contrast, untransduced or GFP-expressing cells treated with TGFb ligand (10 ng/mL) exhibited no significant changes in cell viability (Supplementary Fig. S3 A, B) or neuronal network density (Fig. 5 E, Supplementary Fig. S3 D, E). To gain further evidence for the role of TGFb signaling in aSyn-induced toxicity, we examined if the increase in cytotoxicity after TGFb treatment could be mitigated by knocking down key genes in the TGFb signaling pathway. Therefore, aSyn-overexpressing LUHMES cells were treated with TGFb1, TGFb2, or TGFb3 and transfected with siPOOL siRNAs against ALK5 , SMAD2 , and DYNLRB1 . As expected, the knockdown of ALK5 , SMAD2 , and DYNLRB1 effectively ameliorated the increase in toxicity resulted from the treatment with TGFb ligands. In TGFb1-treated aSyn-overexpressing cells, a reduction in LDH release was observed upon the knockdown of ALK5 (67.7 ± 2.6%, p < 0.001), SMAD2 (86.6 ± 3.0%, p < 0.001), and DYNLRB1 (86.4 ± 2.2%, p < 0.001), in comparison to the LDH released from TGFb1-treated aSyn-overexpressing cells (113.3 ± 2.0%; Fig. 5 A). Similarly, in TGFb2-treated aSyn-overexpressing cells, a reduction in LDH release was observed upon the knockdown of ALK5 (67.8 ± 4.3%, p < 0.001), SMAD2 (89.8 ± 1.3%, p < 0.001), and DYNLRB1 (86.6 ± 4.3%, p < 0.001), in comparison to the LDH released from TGFb2-treated aSyn-overexpressing cells (114.4 ± 2.2%; Fig. 5 B). The same trend was also observed in TGFb3-treated aSyn-overexpressing cells, where a reduction in LDH release was observed upon the knockdown of ALK5 (67.5 ± 1.6%, p < 0.001), SMAD2 (89.5 ± 2.5%, p < 0.001) and DYNLRB1 (86.9 ± 2.3%, p < 0.001), in comparison to TGFb3-treated aSyn-overexpressing cells (112.6 ± 2.5%; Fig. 5 C). The decrease in cytotoxicity resulted from the knockdown of ALK5 , SMAD2 , and DYNLRB1 was also reflected in the increase in the density of neuronal network of TGFb1-treated aSyn-overexpressing cells (Fig. 5 D, E). Specifically, the knockdown of ALK5 in TGFb1-treated aSyn-overexpressing cells led to a significant increase in the number of quadruple points per cell from 72.9 ± 7.2 to 147.4 ± 8.0 ( p < 0.001), the knockdown of SMAD2 to 161.9 ± 9.6 ( p < 0.001), and the knockdown of DYNLRB1 to 111.9 ± 7.5 ( p = 0.02). In summary, with these experiments, we demonstrated that an increase in TGFb signaling via treatment with TGFb ligands exacerbates toxicity in aSyn-overexpressing cells, and this detrimental effect can be mitigated through the knockdown of ALK5 , SMAD2 , and DYNLRB1 . In addition, we also performed a staining of activated caspases 3/7 as apoptotic marker to complement our findings (Supplementary Fig. S4A, B). Even though TGFb1 treatment in aSyn-overexpressing cells did not lead to a significant increase in the intensity of caspase 3/7 signals as compared to untreated aSyn-overexpressing cells (100 ± 7.3%), a significant decrease in caspase 3/7 signals was observed upon the knockdown of ALK5 (65.0 ± 3.4%), SMAD2 (57.8 ± 6.8%), or DYNLRB1 (77.3 ± 1.3%), indicating a significant decrease in the number of apoptotic cells. These results further illustrate that the knockdown of TGFb signaling can effectively counteract the increase of aSyn-induced toxicity resulting from TGFb1 treatment. To further understand why TGFb ligand treatment exacerbated aSyn-induced toxicity, we examined if TGFb ligand treatment influenced SNCA expression. Interestingly, we observed that TGFb ligand treatment led to an additional up-regulation of the SNCA mRNA in aSyn-overexpressing cells (untreated aSyn-overexpressing cells: 24.0 ± 2.0; TGFb1 treatment: 29.5 ± 2.9, p = 0.07; TGFb2 treatment: 33.7 ± 3.0, p < 0.001; TGFb3 treatment: 34.1 ± 3.8, p < 0.001; Fig. 5 F). In contrast, this treatment did not change the expression of the SNCA in untransduced cells or GFP-expressing cells. To determine if TGFb ligand treatment had an influence on the transcriptional activities of the adenoviral vectors, we then investigated if TGFb ligand treatment would also lead to an up-regulation of GFP in GFP-expressing cells. However, there was no significant increase in the expression of the GFP transcript following treatment with TGFb1, TGFb2, or TGFb3 in GFP-expressing cells (Supplementary Fig. S3 C). These findings indicate that the up-regulation of SNCA induced by TGFb ligand treatment was specific to the aSyn-overexpressing condition and not due to an increase of the transcriptional activities of the adenoviral vectors. Transcripts of the TGFb pathway were up-regulated upon aSyn-overexpression Next, we examined if the overexpression of aSyn led to changes in the expression of genes involved in the TGFb pathway. We have previously identified a significant increase in the expression of SMAD2 (1.3 ± 0.1 fold, p = 0.05; Supplementary Fig. S1 C) and SMAD3 (4.6 ± 0.1 fold, p < 0.001, Supplementary Fig. S1 D) in aSyn-overexpressing cells. In contrast, we did not observe an up-regulation of ALK4 or ALK5 in aSyn-overexpressing cells (Fig. 3 ). To expand upon these findings, we analyzed the expression levels of four additional key TGFb pathway genes, namely TGFB1 , TGFB2 , TGFB3 , and TGFBR2 in untransduced cells, aSyn-overexpressing cells, and GFP-expressing cells (Fig. 6 ). We found that aSyn-overexpressing cells, in comparison to untransduced cells, showed higher expression levels of TGFB1 (1.6 ± 0.1 fold, p = 0.001) and TGFB2 (3.8 ± 0.3 fold, p < 0.001). Interestingly, the expressions of TGFB1 , TGFB2 , TGFB3 , and TGFBR2 in untransduced LUHMES cells were comparable (between 28th to 32nd threshold cycle, Supplementary Fig. S3 F). On the other hand, we did not observe any differences in the expression of genes of the TGFb pathway between untransduced cells and GFP-expressing cells (Fig. 6 , Supplementary Fig. S3 G). This suggests that TGFb signaling was up-regulated because of aSyn overexpression. Discussion Previously, in a genome-wide siRNA screening of a PD cell model, we identified that the knockdown of three genes ( ALK7 , DYNLRB1 , and GSK3B ), which are associated with the TGFb signaling pathway, was found to mitigate aSyn-induced toxicity (Höllerhage et al., 2025). Hence, in this project, we investigated the role of the TGFb signaling pathway in aSyn-induced toxicity. Consistent with our initial findings in the siRNA screening, the knockdown of DYNLRB1 and GSK3B by different knockdown paradigms (i.e. esiRNA and siPOOL siRNA) protected human dopaminergic neuron-like cells (LUHMES cells) from aSyn-induced toxicity. In addition, the treatment with two inhibitors of GSK3B, tideglusib and GSK8, also demonstrated a protective effect. On the other hand, while we identified that the transfection of the esiRNA provided protection against aSyn-induced toxicity, the siPOOL siRNA against ALK7 did not. A deeper analysis revealed that the actual target of the ALK7 esiRNA was ALK5 , the gene coding for type-I receptor of the TGFb pathway. We subsequently proved that the knockdown of ALK5 safeguarded LUHMES cells from aSyn-induced toxicity. Furthermore, treatment with a specific ALK5 inhibitor (SM16) also confered a similar protective effect. In addition to ALK5 , we identified that the knockdown of SMAD2 , a downstream target of ALK5, also protected against aSyn-induced toxicity. In addition to this demonstration that the reduction of TGFb signaling was protective against aSyn-induced toxicity, we found that the opposite (i.e. stimulation of TGFb signaling by ligand treatment) was detrimental in our PD cell model. Furthermore, the knockdown of TGFb-associated genes abolished the adverse effects evoked by TGFb ligands. Moreover, we found that TGFb ligand treatment led to an increase in SNCA expression in our PD cell model, and aSyn-overexpression led to an up-regulation of TGFB1 , TGFB2 , SMAD2 , and SMAD3 in LUHMES cells. In this project, we have provided compelling evidence that the reduction of TGFb signaling protected dopaminergic neurons from aSyn-induced toxicity, and an increase in TGFb signaling led to an elevation of aSyn-induced toxicity. Even though TGFb ligands were reported to be generally neurotrophic to dopaminergic neurons [ 24 ], few studies have explored the therapeutic potential of the TGFb signaling pathway in PD. In these toxin-based PD models, mouse or rat dopaminergic primary neurons or mice were treated with a mitochondrial complex I inhibitor MPP + or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; precursor of MPP + ) that specifically kills dopaminergic neurons [ 39 ]. However, the outcomes varied. On one hand, one study reported that dopaminergic neurons in rat mesencephalic primary cultures that had been treated with TGFb1 were less susceptible to MPP + [ 24 ]. In line with that, another study showed that an increase in TGFb signaling via the expression of a constitutively active ALK5 protected mouse dopaminergic neurons in the SN from MPTP-induced toxicity in vivo [ 26 ]. On the other hand, one study reported that the adenoviral overexpression of TGFb1 in the striatum led to a decrease in the number of midbrain dopaminergic neurons in MPTP-injected mice in vivo [ 25 ]. These conflicting outcomes highlight the uncertainty surrounding the role of TGFb signaling in toxin-based PD models. In distinction from previous studies, we investigated the role of TGFb signaling in a human PD cell model that shows toxicity induced by human aSyn. One advantage of our cell model is its close resemblance to neurons that degenerate in PD, providing a more accurate representation of the disease in humans. One study demonstrated that aSyn-toxicity models could replicate human PD pathology more closely than toxin models like 6-hydroxydopamine (6-OHDA) in vivo . The overexpression of aSyn resulted in a progressive loss of dopaminergic neurons that occurred in the course of eight weeks, accompanied by axonal swellings and an impairment in post-synaptic activity. These features were absent in 6-OHDA models [ 40 ]. Our human aSyn toxicity cell model enabled us to investigate how changes in the TGFb signaling led to changes in aSyn-induced cytotoxicity, as well as to observe how aSyn and the genes/proteins of the TGFb pathway could modulate the expression of each other. Importantly, we found that TGFb ligand treatment induced an up-regulation of the SNCA gene upon aSyn overexpression. Similar contradictions in neuroprotection were observed with glial cell-derived neurotrophic factor (GDNF), another member of the TGFb ligand family. While GDNF treatment was protective against MPTP-induced toxicity in mice and monkey in vivo [ 41 , 42 ], it failed to protect dopaminergic neurons against aSyn-induced toxicity in rats in vivo [ 43 ]. Furthermore, a recent clinical trial in which GDNF was infused into the putamen of PD patients via a skull-mounted transcutaneous port and four separate intraputamental infusion catheters also failed to meet its primary endpoint, as GDNF treatment did not lead to significant improvement of the motor symptoms in comparison to placebo control [ 44 ]. These results underscore the critical importance of selecting appropriate experimental models, as differences in experimental models could result in entirely opposite conclusions. This study employed three distinct measurements to assess cell viability/cell death to verify whether the inhibition of TGFb signaling in aSyn-overexpressing cells could provide protection against aSyn-induced toxicity. Results from the LDH release assay and the network analysis indicated that suppressing TGFb signaling significantly reduced cell death in aSyn overexpressing cells and simultaneously led to a significant increase in the density of neuronal network. Furthermore, staining for activated caspases 3/7 confirmed the protective effect of reducing TGFb signaling. In TGFb1-treated aSyn-overexpressing cells, the knockdown of ALK5 , SMAD2 , and DYNLRB1 all led to a significant reduction in the caspase signals. Even though all three assays assess cell death or cell viability, they differ fundamentally. LDH assay measures LDH released into the culture medium, which reflects membrane integrity; the network analysis is an indicator for neuronal network complexity; and the staining for activated caspases 3/7 serves as an apoptotic marker. These methodological differences between the assays could account for the differences in results. Nonetheless, our findings highlight SMAD2 can be a promising therapeutic against PD, since knockdown of SMAD2 showed pronounced protective efficacy consistently across all three readout methods. In this project, we identified an up-regulation of TGFB1 , TGFB2 , SMAD2 , and SMAD3 as consequence of aSyn-overexpression in LUHMES cells. In accordance with this, it has been previously shown that TGFb1 is upregulated in the striatum of post mortem PD patients [ 45 ]. Furthermore, it has been reported previously that TGFb2 was elevated in the vCSF of PD patients in comparison to healthy controls [ 46 ]. The up-regulation of genes and proteins of the TGFb pathway in multiple regions of the brain could indicate a global up-regulation in the brains of PD patients. However, the expression patterns of the genes and proteins of the TGFb pathway in the brains of PD patients are not well understood. In particular, it is unknown whether such up-regulation follows the spread of aSyn pathology. Furthermore, the time point at which such an up-regulation could occur is unknown. To understand if the up-regulation of genes and proteins of the TGFb pathway in the brains of PD patients is global and progressive, deeper analyses of various brain regions would be needed, which was beyond the scope of this study. Our results offer valuable insights for the development of potential drugs against PD. As an example, we identified two inhibitors of GSK3B and one inhibitor of ALK5, which significantly protected dopaminergic neurons against aSyn-induced toxicity. Among them, tideglusib has already been used in clinical trials in patients with progressive supranuclear palsy [ 47 ] and Alzheimer’s disease [ 48 ], meaning existing data on its dosage and safety are already available. In the context of PD, tideglusib treatment protected dopaminergic neurons in mice against MPP + -induced toxicity [ 49 ]. Together with our findings that tideglusib protected dopaminergic neurons from aSyn-induced toxicity, it would be worthwhile to continue with clinical trials of tideglusib in PD. On the other hand, SM16 has been used in vivo in rodents to inhibit tumor growth, and therefore its safety in vivo has also been proven [ 50 ]. However, SM16 has never been used in clinical trials or in experiments that involve non-human primates in vivo . As our results provided clear indications that the use of SM16 strongly protected dopaminergic neurons from aSyn-induced toxicity in vitro , it would be worthwhile to assess if SM16 also exerts its protectiveness against aSyn-induced toxicity in vivo [ 51 ]. In summary, our findings highlight the detrimental role of TGFb signaling in relation to aSyn-induced toxicity. Furthermore, we observed that the reduction of TGFb signaling protected against aSyn-induced toxicity. Our results also suggest that SNCA expression and TGFb pathway gene expression may influence each other. The alignment between our in vitro results and the published observations in human post mortem brains as well as vCSF of PD patients [ 45 , 46 ] support our findings and thus could be the basis for the development of TGFb inhibiting drugs as therapeutic approach for PD and related synucleinopathies. Materials and Methods Cell Culture LUHMES cells (CRL-2927, American Type Culture Collection), were used in all in vitro experiments. Cells were kept at 37°C, 5% CO 2 , and moisturized air. The cell culture flasks (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were pre-coated at 37°C overnight with 0.1 mg/mL poly-L-ornithine (PLO; Sigma-Aldrich, St. Louis, MO, USA). For proliferation, LUHMES cells were maintained in proliferation medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, DMEM/F12 (Sigma-Aldrich) with 1% N2 supplement (Thermo Fisher Scientific), and 0.04 µg/mL fibroblast growth factor-basic, PeproTech, Rocky Hill, CT, USA). For the actual experiments, multi-well plates or flasks (Thermo Fisher Scientific) were pre-coated with 0.1 mg/mL PLO (37°C, overnight) and subsequently with 5 µg/mL fibronectin (FN; R&D Systems, Minneapolis, MN, USA) at 37°C overnight. To initiate the differentiation process, differentiation medium consistent of DMEM/F12 (Sigma-Aldrich) with 1% N2 supplement (Thermo Fisher Scientific), 1 µg/mL tetracycline (Sigma-Aldrich), 0.49 mg/mL dibutyryl-cyclic adenosine monophosphate (Sigma-Aldrich), and 2 ng/µl glial cell line-derived neurotrophic factor (R&D Systems) was used [ 52 ]. Prior the coating with FN, multi-well plates or flasks were washed three times with sterile water to remove excess PLO. Before seeding, the multi-well plates or flasks were washed three times with sterile phosphate buffered saline (PBS; Sigma-Aldrich) to remove excess FN. Cell cultures are routinely tested for mycoplasma to ensure they are not contaminated. Adenoviral transduction On days in vitro (DIV) 2, cells were transduced with adenoviral vectors (Charles Rivers Laboratories, Leiden, the Netherlands) to overexpress human wild-type aSyn or GFP under a cytomegalovirus promotor. 24 hours (h) post-transduction, cells were washed three times with Hank’s Balanced Salt Solution (HBSS; Sigma-Aldrich) to remove residual virus and were then replenished with fresh DM, as previously described [ 52 , 53 ]. siRNA transfection / ligand & inhibitor treatment Approximately 4–6 h after the removal of viral vectors, cells were transfected with siRNAs and/or were treated with ligands or inhibitors. Two different types of siRNAs were used, namely MISSION® endoribonuclease-prepared siRNA (esiRNA, Sigma-Aldrich) and siPOOLs siRNAs (siTOOLs, Martinsried, Germany). Before transfection, the siRNAs were mixed with Opti-MEM™ medium (Thermo Fisher Scientific) and the transfection reagent Lipofectamine™ RNAiMax (Thermo Fisher Scientific). The mixture was then vortexed and incubated for 20 minutes (min) prior to treating the cells. The final concentrations of the esiRNA were 200 ng/µL and those of siPOOL were 5 nM. Ligands or inhibitors were diluted in DM into their respective final concentrations immediately before treating the cells. The following ligands and inhibitors were used: Recombinant human TGFb1 protein (R&D Systems), recombinant human TGFb2 protein (R&D Systems), recombinant human TGFb3 protein (R&D Systems), tideglusib (Sigma-Aldrich), GSK3B-inhibitor VIII (Sigma-Aldrich), or SM16 (Tocris, Minneapolis, MN, USA) Lactate dehydrogenase cytotoxicity assay Lactate dehydrogenase (LDH) as measure for cytotoxicity was quantified by spectrophotometric measurement of the absorbance at 340 nm, which is an indirect measurement of the consumption of nicotinamide adenine dinucleotide (NADH) by LDH. Briefly, culture medium was harvested from cells on DIV8, and was mixed with a reaction buffer consistent of 74.24 mM Tris/HCl (Sigma-Aldrich), 185.6 mM NaCl (Sigma-Aldrich), 3.2 mM pyruvate (Sigma-Aldrich), and 4 mM NADH (Sigma-Aldrich) in water. We then measured the absorbance of the mixture at 340 nm and 420 nm with the use of a plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). Medium from cells lysed with 10% Triton X-100 (Sigma-Aldrich) 1 h before the assay was used as positive control. Unless otherwise specified, all LDH data were normalized to the LDH measured in aSyn-overexpressing cells. Real-time quantitative polymerase chain reaction We used RNeasy® Mini kit (Qiagen, Hilden, Germany) for the extraction of RNA. Briefly, we harvested cells on DIV6 using RLT buffer (activated with 1% beta-mercaptoethanol; Sigma-Aldrich) and cell scrapers. Then, the samples were run through genomic DNA eliminator columns. The resultant flow-through was mixed thoroughly with 70% ethanol, and the mixture was eluded through RNA spin-columns. RNase-free water was used to dissolve the RNA extracted in the RNA spin-columns. RNA concentration was determined using a NanoDrop™ 2000 Microvolume Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). A CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories) was used for the real-time quantitative polymerase chain reaction (qPCR) and SYBR™ Select Master Mix for CFX (Thermo Fisher Scientific) was used as a dye to indicate double-stranded DNA formed in the qPCR process. Western blotting Cells were harvested on DIV6 using M-PER™ lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). The lysates were then centrifuged at 4°C, 10 min, 13,000 g to remove cell debris. The concentration of each lysate was then determined by performing the bicinchoninic acid (BCA) assay using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific). Then, protein samples were loaded on 4–12% Criterion™ XT bis-tris precast protein gels (Bio-Rad Laboratories) with 2-(N-morpholino)ethanesulfonic acid (MES) running buffer. After the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the gels were transferred onto pre-assembled 0.2 µm polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories) in a Trans-Blot® SD semi-dry blotter (Bio-Rad Laboratories). After the transfer, membranes were incubated with 0.4% paraformaldehyde (PFA; Sigma-Aldrich) for 30 min. The membranes were then rinsed with PBS three times (5 min each), followed by an incubation in blocking solution (30% Roti®Block (Carl Roth, Karlsruhe, Germany)) in Tris-Buffered Saline with 0.1% Tween 20 (TBST; Sigma-Aldrich) for 1 h at room temperature (RT). Then, the membranes were incubated with primary antibodies diluted with 10% Roti®Block (Carl Roth)/TBST solution at 4°C overnight. On the next day, membranes were rinsed three times with TBST (5 min each) to remove the primary antibodies and then were incubated with the secondary antibodies (diluted in TBST) for 2 h at RT. After the incubation with the secondary antibodies, the membranes were rinsed three times with TBST (5 min each) to remove the secondary antibodies. Then, the membranes were incubated with Clarity™ Western ECL Substrate (Bio-Rad Laboratories). An Odyssey® Fc Imager (LI-COR Biotechnology, Lincoln, NE, USA) was used to obtain images of the membranes. The following primary antibodies were used: rabbit anti-aSyn (1:500; 701085, Thermo Fisher Scientific); rabbit anti-GAPDH (1:1000; 2118, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-GSK3B (1:5000; ab32391, Abcam, Cambridge, UK). The following secondary antibodies were used: HRP-coupled goat anti-mouse IgG (1:5000; PI-2000, Vector Laboratories, Burlingame, CA, USA); HRP-coupled goat anti-rabbit IgG (1:5000; PI-1000, Vector Laboratories). Immunocytochemistry Prior the start of experiments, glass cover slips (Bellco Glass Inc., Vineland, NJ, USA) were coated in 24-well plates (Thermo Fisher Scientific) with PLO and FN before seeding the cells. The coating procedures and the cell culture were performed as described above. On DIV8, cells were washed two times with HBSS before fixation with 4% PFA in PBS. The fixation was performed at RT for 45 min. Then, the cells were incubated with 0.1% Triton X-100 (Sigma-Aldrich) solution for 15 min at RT. Afterwards, cells were washed three times with PBS, followed by an incubation in blocking solution (5% normal horse serum in PBS) for 1 h at RT. Then, primary antibodies, diluted in blocking solution, were added to the cells followed by an incubation overnight at 4°C. The primary antibodies were removed on the next day and the cells were washed three times with PBS. Then, the cells were incubated with the secondary antibodies (diluted in blocking solution) for 2 h at RT in dark to prevent photobleaching. The cells were then washed once with PBS, and were subsequently incubated with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, diluted with PBS at a concentration of 1:10,000) in dark for 20 min at RT. Before mounting, cells were washed three times with PBS to remove all the antibodies and DAPI. Mowiol® (Carl Roth) was used as mounting medium. Images were acquired with the use of a Leica DMi8 fluorescence microscope (Leica, Wetzlar, Germany) with 40x magnification. Images were processed using the Leica Application Suite X software. To quantify the density of the neuronal network, we counted the number of quadruple points per cell by using a modified Neurite Analyzer plugin on the Fiji software [ 54 ]. Each quadruple point is defined as a neuronal junction that contained, or was connected to four different neurites. The following primary antibodies were used: rabbit anti-aSyn (1:1000; 701085, Thermo Fisher Scientific); mouse anti beta (III)-tubulin (1:1000; 801202, BioLegend, San Diego, CA, USA). The following secondary antibodies were used: Alexa Fluor 555 Donkey anti-rabbit IgG (1:500; A31572, Thermo Fisher Scientific); Alexa Fluor 647 Donkey anti-mouse IgG (1:500; A32794, Thermo Fisher Scientific). Detection of activated caspase-3/7 in apoptotic cells CellEvent™ Caspase-3/7 Detection Reagent (Thermo Fisher) was used to detect the activation of caspase 3/7 in apoptotic cells. Briefly, the cell culture was performed as mentioned previously. On DIV8, prior the assay, the cell culture medium was removed from the cells. CellEvent™ reagent was diluted with pre-warmed HBSS to a final working concentration of 2 µM. The diluted CellEvent™ reagent was added to the cells in a dropwise manner to avoid the cell layer from detaching. The cells were incubated with the reagent for 30 min in the cell culture incubator (37°C, 5% CO 2 ).. After 30 min, the diluted CellEvent™ solution was replaced by diluted Hoechst /HBSS solution (final concentration: 1:10000) and was incubated in the cell culture incubator for 5 min. Imaging was performed with a DMi8 live cell imaging fluorescence microcope. The images were processed using the Leica Application Suite X software. The activated caspases signal in the area of the nuclei was quantified by using the Fiji software. Statistical analysis Data were tested for normality and were analyzed by GraphPad Prism (version 10, GraphPad Software, La Jolla, CA, USA). In all experiments, a minimum of three individual biological replicates were performed. Data were analyzed by multiple t-test if only two experimental groups were present, or by analysis of variance (ANOVA) followed by a post-hoc Bonferroni’s test, if at least three experimental groups were present. Statistical significance was assumed if p values were lower than 0.05. All graphs are presented with error bars indicating the standard error of the mean (SEM). Investigators were not blinded to group allocation during the experiments or outcome assessments. No statistical method was employed to predetermine sample size; instead, sample sizes were determined empirically based on prior experience and observed experimental variability. Abbreviations 6-OHDA: 6-hydroxydopamine ACVR1C: Activin A receptor type 1C ALK4: Activin receptor-like kinase 4 ALK5: Activin receptor-like kinase 5 ALK7: Activin receptor-like kinase 7 ANOVA: analysis of variance aSyn: alpha-synuclein BCA: bicinchoninic acid DAPI: 4',6-diamidino-2-phenylindole DIV: days in vitro DYNLRB1: Dynein light chain roadblock type-1 esiRNA: endoribonuclease-prepared siRNA GAPDH: Glyceraldehyde 3-phosphate dehydrogenase GDNF: Glial cell-derived neurotrophic factor GFP: Green fluorescent protein GSK3B: Glycogen synthase kinase 3 beta GSK8: GSK3B inhibitor-VIII HBSS: Hank’s Balanced Salt Solution huUBQLN1: human ubiquilin 1 LBs: Lewy bodies LDH: lactate dehydrogenase LUHMES cells: Lund Mesencephalic cells MES: 2-(N-morpholino)ethanesulfonic acid MPP + : 1-methyl-4-phenylpyridinium MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine NADH: nicotinamide adenine dinucleotide PBS: phosphate buffered saline PD: Parkinson’s disease PFA: paraformaldehyde POLR2A: RNA Polymerase II Subunit A PVDF: polyvinylidene fluoride SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis SMAD2: Mothers against decapentaplegic homolog 2 SMAD3: Mothers against decapentaplegic homolog 3 SMAD4: Mothers against decapentaplegic homolog 4 SN: substantia nigra TBST: Tris-Buffered Saline with 0.1% Tween 20 TGFb: Transforming Growth Factor-beta TGFBR1: TGFb type-I receptor TGFBR2: TGFb type-II receptor TH: Tyrosine hydroxylase vCSF: ventricular cerebrospinal fluid Declarations Ethics approval and consent to participate : N/A Consent for publication : N/A Availability of data and materials : All datasets used and analyzed in this study are available from the corresponding authors on reasonable request. Competing interest : All authors have no competing interests. Funding : This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy – ID 390857198) and within the Hannover Cluster RESIST (EXC 2155 – - project number 39087428), the German Federal Ministry of Education and Research (BMBF, 01KU1403A EpiPD); the ParkinsonFonds Germany (Hypothesis-free compound screen, alpha-Synuclein fragments in PD); Deutsche Forschungsgemeinschaft (DFG, HO2402/18-1 MSAomics); VolkswagenStiftung (Niedersächsisches Vorab); Petermax-Müller Foundation (Etiology and Therapy of Synucleinopathies and Tauopathies). Authors' contributions : OWC and MH designed the experiments. OWC, LD, SB, VE, and CM performed the experiments. OWC and MH analyzed the data. OWC created the figures and tables and wrote the first draft of the manuscript. All authors critically revised the manuscript. MB, GUH, and MH conceived the study. GUH and MH provided overall project leadership. All authors discussed and commented on the manuscript and agreed to publication. Acknowledgements : We thank Sabine Lang (MHH, Dept. for Neurology) for technical support. References Aragon, E., Wang, Q., Zou, Y., Morgani, S. M., Ruiz, L., Kaczmarska, Z., Su, J., Torner, C., Tian, L., Hu, J., Shu, W., Agrawal, S., Gomes, T., Marquez, J. A., Hadjantonakis, A. K., Macias, M. J., & Massague, J. (2019). Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-beta signaling. Genes Dev , 33 (21-22), 1506-1524. https://doi.org/10.1101/gad.330837.119 Chakroun, T., Evsyukov, V., Nykanen, N. P., Hollerhage, M., Schmidt, A., Kamp, F., Ruf, V. C., Wurst, W., Rosler, T. W., & Hoglinger, G. U. (2020). Alpha-synuclein fragments trigger distinct aggregation pathways. Cell Death Dis , 11 (2), 84. https://doi.org/10.1038/s41419-020-2285-7 Chia, S. J., Tan, E. K., & Chao, Y. X. (2020). Historical Perspective: Models of Parkinson's Disease. Int J Mol Sci , 21 (7). https://doi.org/10.3390/ijms21072464 Decressac, M., Mattsson, B., & Bjorklund, A. (2012). Comparison of the behavioural and histological characteristics of the 6-OHDA and alpha-synuclein rat models of Parkinson's disease. Exp Neurol , 235 (1), 306-315. https://doi.org/10.1016/j.expneurol.2012.02.012 Decressac, M., Ulusoy, A., Mattsson, B., Georgievska, B., Romero-Ramos, M., Kirik, D., & Bjorklund, A. (2011). GDNF fails to exert neuroprotection in a rat alpha-synuclein model of Parkinson's disease. Brain , 134 (Pt 8), 2302-2311. https://doi.org/10.1093/brain/awr149 Dobolyi, A., Vincze, C., Pal, G., & Lovas, G. (2012). The neuroprotective functions of transforming growth factor beta proteins. Int J Mol Sci , 13 (7), 8219-8258. https://doi.org/10.3390/ijms13078219 Dong, Y., Lu, J., Zhang, S., Chen, L., Wen, J., Wang, F., Mao, Y., Li, L., Zhang, J., Liao, S., & Dong, L. (2023). Design, synthesis and bioevaluation of 1,2,4-thiadiazolidine-3,5-dione derivatives as potential GSK-3beta inhibitors for the treatment of Alzheimer's disease. Bioorg Chem , 134 , 106446. https://doi.org/10.1016/j.bioorg.2023.106446 Emin, D., Zhang, Y. P., Lobanova, E., Miller, A., Li, X., Xia, Z., Dakin, H., Sideris, D. I., Lam, J. Y. L., Ranasinghe, R. T., Kouli, A., Zhao, Y., De, S., Knowles, T. P. J., Vendruscolo, M., Ruggeri, F. S., Aigbirhio, F. I., Williams-Gray, C. H., & Klenerman, D. (2022). Small soluble alpha-synuclein aggregates are the toxic species in Parkinson's disease. Nat Commun , 13 (1), 5512. https://doi.org/10.1038/s41467-022-33252-6 Engebretsen, K. V., Skardal, K., Bjornstad, S., Marstein, H. S., Skrbic, B., Sjaastad, I., Christensen, G., Bjornstad, J. L., & Tonnessen, T. (2014). Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5. J Mol Cell Cardiol , 76 , 148-157. https://doi.org/10.1016/j.yjmcc.2014.08.008 Gash, D. M., Zhang, Z., Ovadia, A., Cass, W. A., Yi, A., Simmerman, L., Russell, D., Martin, D., Lapchak, P. A., Collins, F., Hoffer, B. J., & Gerhardt, G. A. (1996). Functional recovery in parkinsonian monkeys treated with GDNF. Nature , 380 (6571), 252-255. https://doi.org/10.1038/380252a0 Guo, X., Ramirez, A., Waddell, D. S., Li, Z., Liu, X., & Wang, X. F. (2008). Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling. Genes Dev , 22 (1), 106-120. https://doi.org/10.1101/gad.1590908 Haas, A. J., Prigent, S., Dutertre, S., Le Drean, Y., & Le Page, Y. (2016). Neurite analyzer: An original Fiji plugin for quantification of neuritogenesis in two-dimensional images. J Neurosci Methods , 271 , 86-91. https://doi.org/10.1016/j.jneumeth.2016.07.011 Hata, A., & Chen, Y. G. (2016). TGF-beta Signaling from Receptors to Smads. Cold Spring Harb Perspect Biol , 8 (9). https://doi.org/10.1101/cshperspect.a022061 Heldin, C. H., & Moustakas, A. (2016). Signaling Receptors for TGF-beta Family Members. Cold Spring Harb Perspect Biol , 8 (8). https://doi.org/10.1101/cshperspect.a022053 Hill, C. S. (2016). Transcriptional Control by the SMADs. Cold Spring Harb Perspect Biol , 8 (10). https://doi.org/10.1101/cshperspect.a022079 Hoglinger, G. U., Adler, C. H., Berg, D., Klein, C., Outeiro, T. F., Poewe, W., Postuma, R., Stoessl, A. J., & Lang, A. E. (2024). A biological classification of Parkinson's disease: the SynNeurGe research diagnostic criteria. Lancet Neurol , 23 (2), 191-204. https://doi.org/10.1016/S1474-4422(23)00404-0 Hoglinger, G. U., Huppertz, H. J., Wagenpfeil, S., Andres, M. V., Belloch, V., Leon, T., Del Ser, T., & Investigators, T. M. (2014). Tideglusib reduces progression of brain atrophy in progressive supranuclear palsy in a randomized trial. Mov Disord , 29 (4), 479-487. https://doi.org/10.1002/mds.25815 Höllerhage, M., Duan, L., Chua, OWH., Moebius, C., Bothe, S., Hopfner, F., Wahl-Schott., C., Bickle., M., Höglinger., GU. (2025). A genome-wide RNA interference screening reveals protectiveness of SNX5 knockdown in a Parkinson’s disease cell model. Transl Neurodegener. 2025 Jun 3;14(1):27. https://doi.org/10.1186/s40035-025-00486-5. Hollerhage, M., Goebel, J. N., de Andrade, A., Hildebrandt, T., Dolga, A., Culmsee, C., Oertel, W. H., Hengerer, B., & Hoglinger, G. U. (2014). Trifluoperazine rescues human dopaminergic cells from wild-type alpha-synuclein-induced toxicity. Neurobiol Aging , 35 (7), 1700-1711. https://doi.org/10.1016/j.neurobiolaging.2014.01.027 Hollerhage, M., Stepath, M., Kohl, M., Pfeiffer, K., Chua, O. W. H., Duan, L., Hopfner, F., Eisenacher, M., Marcus, K., & Hoglinger, G. U. (2022). Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein. Front Neurol , 13 , 787059. https://doi.org/10.3389/fneur.2022.787059 Jin, Q., Ding, W., & Mulder, K. M. (2007). Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway. J Biol Chem , 282 (26), 19122-19132. https://doi.org/10.1074/jbc.M609915200 Jornvall, H., Blokzijl, A., ten Dijke, P., & Ibanez, C. F. (2001). The orphan receptor serine/threonine kinase ALK7 signals arrest of proliferation and morphological differentiation in a neuronal cell line. J Biol Chem , 276 (7), 5140-5146. https://doi.org/10.1074/jbc.M005200200 Jornvall, H., Reissmann, E., Andersson, O., Mehrkash, M., & Ibanez, C. F. (2004). ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse. Mol Cell Biol , 24 (21), 9383-9389. https://doi.org/10.1128/MCB.24.21.9383-9389.2004 Kang, H., Seo, E., Oh, Y. S., & Jun, H. S. (2022). TGF-beta activates NLRP3 inflammasome by an autocrine production of TGF-beta in LX-2 human hepatic stellate cells. Mol Cell Biochem , 477 (5), 1329-1338. https://doi.org/10.1007/s11010-022-04369-5 Komai, T., Inoue, M., Okamura, T., Morita, K., Iwasaki, Y., Sumitomo, S., Shoda, H., Yamamoto, K., & Fujio, K. (2018). Transforming Growth Factor-beta and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals. Front Immunol , 9 , 1364. https://doi.org/10.3389/fimmu.2018.01364 Krieglstein, K. (2004). Factors promoting survival of mesencephalic dopaminergic neurons. Cell Tissue Res , 318 (1), 73-80. https://doi.org/10.1007/s00441-004-0920-8 Krieglstein, K., Miyazono, K., ten Dijke, P., & Unsicker, K. (2012). TGF-beta in aging and disease. Cell Tissue Res , 347 (1), 5-9. https://doi.org/10.1007/s00441-011-1278-3 Krieglstein, K., Suter-Crazzolara, C., Fischer, W. H., & Unsicker, K. (1995). TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J , 14 (4), 736-742. https://doi.org/10.1002/j.1460-2075.1995.tb07052.x Krieglstein, K., Suter-Crazzolara, C., & Unsicker, K. (1995). Development of mesencephalic dopaminergic neurons and the transforming growth factor-beta superfamily. J Neural Transm Suppl , 46 , 209-216. https://www.ncbi.nlm.nih.gov/pubmed/8821057 Krieglstein, K., Zheng, F., Unsicker, K., & Alzheimer, C. (2011). More than being protective: functional roles for TGF-beta/activin signaling pathways at central synapses. Trends Neurosci , 34 (8), 421-429. https://doi.org/10.1016/j.tins.2011.06.002 Li, J., Ma, S., Chen, J., Hu, K., Li, Y., Zhang, Z., Su, Z., Woodgett, J. R., Li, M., & Huang, Q. (2020). GSK-3beta Contributes to Parkinsonian Dopaminergic Neuron Death: Evidence From Conditional Knockout Mice and Tideglusib. Front Mol Neurosci , 13 , 81. https://doi.org/10.3389/fnmol.2020.00081 Liu, L., Liu, X., Ren, X., Tian, Y., Chen, Z., Xu, X., Du, Y., Jiang, C., Fang, Y., Liu, Z., Fan, B., Zhang, Q., Jin, G., Yang, X., & Zhang, X. (2016). Smad2 and Smad3 have differential sensitivity in relaying TGFbeta signaling and inversely regulate early lineage specification. Sci Rep , 6 , 21602. https://doi.org/10.1038/srep21602 Liu, Q., Qiu, J., Liang, M., Golinski, J., van Leyen, K., Jung, J. E., You, Z., Lo, E. H., Degterev, A., & Whalen, M. J. (2014). Akt and mTOR mediate programmed necrosis in neurons. Cell Death Dis , 5 (2), e1084. https://doi.org/10.1038/cddis.2014.69 Loomans, H. A., & Andl, C. D. (2016). Activin receptor-like kinases: a diverse family playing an important role in cancer. Am J Cancer Res , 6 (11), 2431-2447. https://www.ncbi.nlm.nih.gov/pubmed/27904762 Lotharius, J., Barg, S., Wiekop, P., Lundberg, C., Raymon, H. K., & Brundin, P. (2002). Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J Biol Chem , 277 (41), 38884-38894. https://doi.org/10.1074/jbc.M205518200 Lotharius, J., Falsig, J., van Beek, J., Payne, S., Dringen, R., Brundin, P., & Leist, M. (2005). Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci , 25 (27), 6329-6342. https://doi.org/10.1523/JNEUROSCI.1746-05.2005 Lovestone, S., Boada, M., Dubois, B., Hull, M., Rinne, J. O., Huppertz, H. J., Calero, M., Andres, M. V., Gomez-Carrillo, B., Leon, T., del Ser, T., & investigators, A. (2015). A phase II trial of tideglusib in Alzheimer's disease. J Alzheimers Dis , 45 (1), 75-88. https://doi.org/10.3233/JAD-141959 Mogi, M., Harada, M., Kondo, T., Narabayashi, H., Riederer, P., & Nagatsu, T. (1995). Transforming growth factor-beta 1 levels are elevated in the striatum and in ventricular cerebrospinal fluid in Parkinson's disease. Neurosci Lett , 193 (2), 129-132. https://doi.org/10.1016/0304-3940(95)11686-q Rausch, M. P., Hahn, T., Ramanathapuram, L., Bradley-Dunlop, D., Mahadevan, D., Mercado-Pimentel, M. E., Runyan, R. B., Besselsen, D. G., Zhang, X., Cheung, H. K., Lee, W. C., Ling, L. E., & Akporiaye, E. T. (2009). An orally active small molecule TGF-beta receptor I antagonist inhibits the growth of metastatic murine breast cancer. Anticancer Res , 29 (6), 2099-2109. https://www.ncbi.nlm.nih.gov/pubmed/19528470 Richter, F., Stanojlovic, M., Kaufer, C., Gericke, B., & Feja, M. (2023). A Mouse Model to Test Novel Therapeutics for Parkinson's Disease: an Update on the Thy1-aSyn ("line 61") Mice. Neurotherapeutics . https://doi.org/10.1007/s13311-022-01338-0 Runyan, C. E., Schnaper, H. W., & Poncelet, A. C. (2005). The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells. J Biol Chem , 280 (9), 8300-8308. https://doi.org/10.1074/jbc.M407939200 Sanchez-Capelo, A., Colin, P., Guibert, B., Biguet, N. F., & Mallet, J. (2003). Transforming growth factor beta1 overexpression in the nigrostriatal system increases the dopaminergic deficit of MPTP mice. Mol Cell Neurosci , 23 (4), 614-625. https://doi.org/10.1016/s1044-7431(03)00081-2 Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., & Goedert, M. (1998). alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A , 95 (11), 6469-6473. https://doi.org/10.1073/pnas.95.11.6469 Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., & Goedert, M. (1997). Alpha-synuclein in Lewy bodies. Nature , 388 (6645), 839-840. https://doi.org/10.1038/42166 Suzuki, E., Kim, S., Cheung, H. K., Corbley, M. J., Zhang, X., Sun, L., Shan, F., Singh, J., Lee, W. C., Albelda, S. M., & Ling, L. E. (2007). A novel small-molecule inhibitor of transforming growth factor beta type I receptor kinase (SM16) inhibits murine mesothelioma tumor growth in vivo and prevents tumor recurrence after surgical resection. Cancer Res , 67 (5), 2351-2359. https://doi.org/10.1158/0008-5472.CAN-06-2389 Tesseur, I., Nguyen, A., Chang, B., Li, L., Woodling, N. S., Wyss-Coray, T., & Luo, J. (2017). Deficiency in Neuronal TGF-beta Signaling Leads to Nigrostriatal Degeneration and Activation of TGF-beta Signaling Protects against MPTP Neurotoxicity in Mice. J Neurosci , 37 (17), 4584-4592. https://doi.org/10.1523/JNEUROSCI.2952-16.2017 Tolosa, E., Garrido, A., Scholz, S. W., & Poewe, W. (2021). Challenges in the diagnosis of Parkinson's disease. Lancet Neurol , 20 (5), 385-397. https://doi.org/10.1016/S1474-4422(21)00030-2 Tomac, A., Lindqvist, E., Lin, L. F., Ogren, S. O., Young, D., Hoffer, B. J., & Olson, L. (1995). Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature , 373 (6512), 335-339. https://doi.org/10.1038/373335a0 Tsuchida, K., Sawchenko, P. E., Nishikawa, S., & Vale, W. W. (1996). Molecular cloning of a novel type I receptor serine/threonine kinase for the TGF beta superfamily from rat brain. Mol Cell Neurosci , 7 (6), 467-478. https://doi.org/10.1006/mcne.1996.0034 Tsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L., & Wrana, J. L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell , 95 (6), 779-791. https://doi.org/10.1016/s0092-8674(00)81701-8 Vawter, M. P., Dillon-Carter, O., Tourtellotte, W. W., Carvey, P., & Freed, W. J. (1996). TGFbeta1 and TGFbeta2 concentrations are elevated in Parkinson's disease in ventricular cerebrospinal fluid. Exp Neurol , 142 (2), 313-322. https://doi.org/10.1006/exnr.1996.0200 Whone, A., Luz, M., Boca, M., Woolley, M., Mooney, L., Dharia, S., Broadfoot, J., Cronin, D., Schroers, C., Barua, N. U., Longpre, L., Barclay, C. L., Boiko, C., Johnson, G. A., Fibiger, H. C., Harrison, R., Lewis, O., Pritchard, G., Howell, M., . . . Gill, S. S. (2019). Randomized trial of intermittent intraputamenal glial cell line-derived neurotrophic factor in Parkinson's disease. Brain , 142 (3), 512-525. https://doi.org/10.1093/brain/awz023 Wong, Y. C., & Krainc, D. (2017). alpha-synuclein toxicity in neurodegeneration: mechanism and therapeutic strategies. Nat Med , 23 (2), 1-13. https://doi.org/10.1038/nm.4269 Zhang, Y., Alexander, P. B., & Wang, X. F. (2017). TGF-beta Family Signaling in the Control of Cell Proliferation and Survival. Cold Spring Harb Perspect Biol , 9 (4). https://doi.org/10.1101/cshperspect.a022145 Additional Declarations (Not answered) Supplementary Files ajchecklistOscar20250611.pdf Reporting summary SupplementaryFile1SuppFigureLegends.docx Figure legends of the supplementary figures SupplementaryFile2uncroppedWB.docx Full Western blot images, uncropped Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Cell Death Discovery → Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-6867964","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475797100,"identity":"492d7b1f-e012-498b-b7ab-215a7e13293c","order_by":0,"name":"Matthias Höllerhage","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-5333-0121","institution":"MHH","correspondingAuthor":true,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Höllerhage","suffix":""},{"id":475797101,"identity":"f551e62a-cf73-4d4e-8625-7811600bdfbf","order_by":1,"name":"Oscar Wing Ho Chua","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"Wing Ho","lastName":"Chua","suffix":""},{"id":475797102,"identity":"0eaf14db-0c15-474d-ae6e-c5d2bcb85298","order_by":2,"name":"Linghan Duan","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Linghan","middleName":"","lastName":"Duan","suffix":""},{"id":475797103,"identity":"7bd166e0-a910-4711-a6bc-0ba437d77224","order_by":3,"name":"Svenja Bothe","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Svenja","middleName":"","lastName":"Bothe","suffix":""},{"id":475797104,"identity":"b5abe180-6a82-41b5-959e-44c7994cc4a2","order_by":4,"name":"Valentin Evsyukov","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Valentin","middleName":"","lastName":"Evsyukov","suffix":""},{"id":475797105,"identity":"a64814af-232a-4c55-8e2e-77af69b7d7b7","order_by":5,"name":"Claudia Moebius","email":"","orcid":"","institution":"Max Planck Institute of Molecular Cell Biology and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Moebius","suffix":""},{"id":475797106,"identity":"295d4f45-5a98-4006-8eb3-3574de4e4b99","order_by":6,"name":"Marc Bickle","email":"","orcid":"","institution":"Max Planck Institute of Molecular Cell Biology and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Bickle","suffix":""},{"id":475797107,"identity":"6d7246de-34d9-4e36-a8db-f5a4a4592099","order_by":7,"name":"Günter Höglinger","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität Munich","correspondingAuthor":false,"prefix":"","firstName":"Günter","middleName":"","lastName":"Höglinger","suffix":""}],"badges":[],"createdAt":"2025-06-11 05:10:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6867964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6867964/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-025-02901-2","type":"published","date":"2025-12-12T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87666214,"identity":"a062a5e9-75d5-4a08-8057-1e3ea7060f69","added_by":"auto","created_at":"2025-07-27 11:15:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3590171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of the TGF pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon ligand binding (TGFb/Activin/Nodal), respective type-II receptor dimers of the TGFb pathway are activated, recruiting and activating respective type-I receptor dimers. Together they form a heterotetrameric receptor complex that is internalized into the cell. SMAD2 and SMAD3 proteins are presented to the receptor complex via SMAD anchor for receptor activation (SARA). Subsequently, the SMAD2/3 proteins are phosphorylated and are translocated into the nucleus together with SMAD4, where the SMAD complex can bind to various target genes and drive target gene transcription via the transcription factor-binding element (TBE). The TGFb pathway can be regulated by, for example, competitive binding of the inhibitory SMAD7 protein to the receptor complex, or by proteasomal degradation of receptors or SMAD proteins via Smurf1/2 E3 ubiquitin ligases. ALK7 is one of the seven type-I receptors of the TGFb pathway that is specific for the ligand Nodal. DYNLRB1 is a member of the dynein light-chain family that was reported to be closely associated with SMAD2 nuclear translocation. On the other hand, GSK3B has been reported to be involved in the degradation of SMAD3. Created in BioRender. Chua, W. (2022) BioRender.com/e97d354.\u003c/p\u003e","description":"","filename":"ManuscriptFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/c75373726926c9993f6e977e.png"},{"id":87666419,"identity":"c23e7469-4c75-4b29-9cd4-aae40712569d","added_by":"auto","created_at":"2025-07-27 11:23:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3699864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of the genome-wide siRNA screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B:\u003c/strong\u003e Measurement of LDH release into the cell culture medium confirmed the protection against aSyn-induced cytotoxicity by esiRNAs (\u003cstrong\u003eA\u003c/strong\u003e) or siPOOL siRNAs (\u003cstrong\u003eB\u003c/strong\u003e) against \u003cem\u003eGSK3B\u003c/em\u003e, \u003cem\u003eDYNLRB1\u003c/em\u003e, and \u003cem\u003eALK7\u003c/em\u003e. Data are normalized to LDH release of untransfected aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC, D:\u003c/strong\u003e Representative Western blots with an antibody against GSK3B confirmed the knockdown of GSK3B by after transfection with the esiRNA (\u003cstrong\u003eC\u003c/strong\u003e) and the siPOOL siRNA (\u003cstrong\u003eD\u003c/strong\u003e) against \u003cem\u003eGSK3B\u003c/em\u003e in untransduced cells (UTCs) and aSyn-overexpressing cells. Full size blot images of (C) and (D) can be found in Supplementary File Fig. A and B respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE, F:\u003c/strong\u003e The knockdown efficacy of esiRNAs (\u003cstrong\u003eE\u003c/strong\u003e) and siPOOL siRNAs (\u003cstrong\u003eF\u003c/strong\u003e) against \u003cem\u003eDYNLRB1 \u003c/em\u003ewas confirmed by qPCR analysis in UTCs and aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e: The plot shows the number of threshold cycles in the qPCR until which expression of the respective genes was detected in untransduced LUHMES cells. The expressions of \u003cem\u003eALK4\u003c/em\u003e, \u003cem\u003eALK5\u003c/em\u003e, and the two housekeeping genes were comparable (between 24\u003csup\u003eth\u003c/sup\u003e and 27\u003csup\u003eth\u003c/sup\u003e threshold cycle). The expression of \u003cem\u003eALK7\u003c/em\u003e was much lower in comparison to the other four genes in the chart, reaching the threshold value ~10 cycles later than the other four genes.\u003c/p\u003e\n\u003cp\u003eUTCs: untransduced cells; aSyn: cells overexpressing alpha-Synuclein; \u003cem\u003eGSK3B\u003c/em\u003e esiRNA: esiRNA against \u003cem\u003eGSK3B\u003c/em\u003e; \u003cem\u003eDYNLRB1\u003c/em\u003e esiRNA: esiRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e; \u003cem\u003eALK7\u003c/em\u003e esiRNA: esiRNA against \u003cem\u003eALK7\u003c/em\u003e; neg. ctrl. esiRNA: negative control esiRNA targeting firefly luciferase; \u003cem\u003eGSK3B\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eGSK3B\u003c/em\u003e; \u003cem\u003eDYNLRB1\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e; \u003cem\u003eALK7\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e; neg. ctrl. siPOOL: negative control siPOOL siRNA; untransfected: cells that were not transfected with siPOOL siRNAs\u003c/p\u003e\n\u003cp\u003e#: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected UTCs; ^: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ^^^: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against UTCs + neg. ctrl. esiRNA/siPOOL; *: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected aSyn; +++: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against aSyn + neg. ctrl. esiRNA/siPOOL; ns: not significant.\u003c/p\u003e","description":"","filename":"ManuscriptFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/2445d1b006455b6cffd6c801.png"},{"id":87666219,"identity":"49fe5d5d-60d4-4272-a983-0f7df3b29526","added_by":"auto","created_at":"2025-07-27 11:15:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6260371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfirmation of the efficacy of ALK7 knockdown.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B, C\u003c/strong\u003e: Quantification of the relative mRNA expression of \u003cem\u003eALK7\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e); \u003cem\u003eALK4\u003c/em\u003e(\u003cstrong\u003eB\u003c/strong\u003e), and \u003cem\u003eALK5\u003c/em\u003e (\u003cstrong\u003eC\u003c/strong\u003e) after transfection with the esiRNA against \u003cem\u003eALK7\u003c/em\u003e. The esiRNA against \u003cem\u003eALK7\u003c/em\u003e led to a knockdown of \u003cem\u003eALK7\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) and \u003cem\u003eALK5\u003c/em\u003e (\u003cstrong\u003eC\u003c/strong\u003e) only in aSyn-overexpressing cells, but not in untransduced cells (UTCs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD, E, F\u003c/strong\u003e: Quantification of the relative mRNA expression of \u003cem\u003eALK7\u003c/em\u003e (\u003cstrong\u003eD\u003c/strong\u003e); \u003cem\u003eALK4\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e), and \u003cem\u003eALK5\u003c/em\u003e (\u003cstrong\u003eF\u003c/strong\u003e) after transfection with the siPOOL against \u003cem\u003eALK7\u003c/em\u003e. The siPOOL against \u003cem\u003eALK7\u003c/em\u003eled to a knockdown of \u003cem\u003eALK7 \u003c/em\u003eonly in aSyn-overexpressing cells but not in UTCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG, H, I:\u003c/strong\u003eQuantification of the relative mRNA expression of \u003cem\u003eALK7\u003c/em\u003e (\u003cstrong\u003eG\u003c/strong\u003e); \u003cem\u003eALK4\u003c/em\u003e(\u003cstrong\u003eH\u003c/strong\u003e), and \u003cem\u003eALK5\u003c/em\u003e (\u003cstrong\u003eI\u003c/strong\u003e) after transfection with the siPOOL against \u003cem\u003eALK5. \u003c/em\u003eThe siPOOL against \u003cem\u003eALK5\u003c/em\u003e only led to a knockdown of ALK5 in both UTCs and aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003eUTCs: untransduced cells; aSyn: cells overexpressing alpha-Synuclein; \u003cem\u003eALK7\u003c/em\u003eesi: esiRNA against \u003cem\u003eALK7\u003c/em\u003e; neg. ctrl. esiRNA: negative control esiRNA targeting firefly luciferase; \u003cem\u003eALK7\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e; neg. ctrl. siPOOL: negative control siPOOL siRNA; \u003cem\u003eALK5\u003c/em\u003e si: siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e; untransfected: cells that were not transfected with siPOOL siRNAs\u003c/p\u003e\n\u003cp\u003e#: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected UTCs; ^^: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 against UTCs + neg. ctrl. esiRNA/siPOOL; *: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ***: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected aSyn; +: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ++: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, +++: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against aSyn + neg. ctrl. esiRNA/siPOOL; ns: not significant.\u003c/p\u003e","description":"","filename":"ManuscriptFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/53551321a00d2ebbbfe3de72.png"},{"id":87666227,"identity":"0dfa14ae-bd18-4863-9825-96690d63d227","added_by":"auto","created_at":"2025-07-27 11:15:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9837677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of TGFb pathway genes protected LUHMES cells from aSyn-induced toxicity\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Quantification of LDH released into the cell culture medium as measure for cytotoxicity showed that knockdown of \u003cem\u003eALK5\u003c/em\u003eby the siPOOL siRNA protected from aSyn-induced toxicity. In untransduced cells (UTCs) or GFP-expressing cells, the knockdown of \u003cem\u003eALK5\u003c/em\u003e did not change toxicity. Data are normalized to LDH release of untransfected aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB, C\u003c/strong\u003e: Quantification of LDH release into the cell culture medium after treatment of LUHMES cells with SM16, an ALK5 inhibitor. SM16 treatment protected against aSyn-induced toxicity at concentrations of 10 µM, 20 µM, and 40 µM \u003cstrong\u003e(B)\u003c/strong\u003e. In UTCs or GFP-expressing control cells, 20 µM and 40 µM of SM16 had no effect on cell viability (\u003cstrong\u003eC\u003c/strong\u003e). Data are normalized to LDH release of untransfected aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e: Quantification of LDH release into the cell culture medium in UTCs, aSyn-overexpressing, and GFP-expressing cells after transfection with siRNAs against SMAD2 and SMAD3. The knockdown of \u003cem\u003eSMAD2\u003c/em\u003eby siPOOL siRNA protected against aSyn-induced toxicity, whereas the knockdown of \u003cem\u003eSMAD3\u003c/em\u003e did not protect against aSyn-induced toxicity. Data are normalized to LDH release of untransfected aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e: Quantification of the number of quadruple points per cell as a measure for changes in the density of neuronal network showed that aSyn-overexpression led to a reduction in the density of neuronal network. Such reduction could be mitigated by the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e. On the other hand, the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e in UTCs or GFP-expressing cells did not result in changes in the neuronal network complexity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e: Representative images of the ICC staining against DAPI (blue), GFP (green, if any), aSyn (red), and beta-III tubulin (cyan) was performed. Corresponding network analysis for each representative figure is shown below the staining, in which black lines outlined the neurites and the yellow lines outlined the nuclei.\u003c/p\u003e\n\u003cp\u003eUTCs: untransduced cells; aSyn/+aSyn: cells overexpressing alpha-Synuclein; +GFP: cells expressing GFP; \u003cem\u003eALK5\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e; neg. ctrl. siPOOL: negative siPOOL siRNA; \u003cem\u003eSMAD2\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eSMAD2\u003c/em\u003e;\u003cem\u003e SMAD3\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eSMAD3\u003c/em\u003e; untreated: cells that were not treated with SM16; untransfected: cells that were not transfected with siPOOL siRNAs\u003c/p\u003e\n\u003cp\u003e###: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected UTCs; *: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against untransfected aSyn-overexpressing cells. ns: not significant across the compared groups.\u003c/p\u003e","description":"","filename":"ManuscriptFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/857e522b585926085f8cf6aa.png"},{"id":87666225,"identity":"687d016a-eaad-4619-89be-28f46c4e3362","added_by":"auto","created_at":"2025-07-27 11:15:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9930640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStimulation of the TGFb pathway increased toxicity in aSyn-overexpressing cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B, C\u003c/strong\u003e: Quantification of LDH release into the cell culture medium after treatment of LUHMES cells with TGFb1 \u003cstrong\u003e(A)\u003c/strong\u003e, TGFb2 \u003cstrong\u003e(B)\u003c/strong\u003e, or TGFb3 \u003cstrong\u003e(C)\u003c/strong\u003e. The treatment of TGFb ligands led to an increase in aSyn-induced toxicity, as compared to untreated aSyn-overexpressing cells. The knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e by siPOOL siRNA significantly protected TGFb ligand-treated aSyn-overexpressing cells. Data are normalized to LDH release of untreated aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e: Representative images of the ICC staining against DAPI (blue), GFP (green, if any), aSyn (red), and beta-III tubulin (cyan) in aSyn-overexpressing cells. Corresponding network analysis for each representative figure is shown below the staining, in which black lines outlined the neurites and the yellow lines outlined the nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e: Quantification of the number of quadruple points per cell as a measure for changes in the density of neuronal network showed that the treatment of TGFb1 led to a reduction in the density of neuronal network in aSyn-overexpressing cells. The knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e by siPOOL siRNA significantly increased the density of neuronal network in TGFb1-treated aSyn-overexpressing cells. TGFb1 ligand treatment did not result in significant changes in the density of neuronal network in untransduced cells (UTCs) or GFP-expressing cells. In addition, no significant changes in the density of neuronal network was resulted from the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e in TGFb1-treated UTCs or GFP-expressing cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e: Quantification of the relative mRNA expression of \u003cem\u003eSNCA\u003c/em\u003e after the treatment of TGFb1, TGFb2, or TGFb3 in UTCs, aSyn-overexpressing cells, and GFP-expressing cells. TGFb2 and TGFb3 treatment led to a significant increase in the expression of \u003cem\u003eSNCA\u003c/em\u003e only in aSyn-overexpressing cells.\u003c/p\u003e\n\u003cp\u003eUTCs: untransduced cells; aSyn/+aSyn: cells overexpressing alpha-Synuclein; +GFP: cells expressing GFP; \u003cem\u003eALK5\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e; neg. ctrl. siPOOL: negative control siPOOL siRNA; \u003cem\u003eSMAD2\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eSMAD2\u003c/em\u003e; \u003cem\u003eDYNLRB1\u003c/em\u003e siPOOL: siPOOL siRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e; untreated: cells that were not treated with TGFb ligands; untransfected: cells that were not transfected with siPOOL siRNAs\u003c/p\u003e\n\u003cp\u003e#: \u003cem\u003ep \u003c/em\u003e\u0026lt;0.05, ###: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001 against untreated UTCs; *: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001 against untreated aSyn; §: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, §§§: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001 against aSyn+TGFb ligand; ^: \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 against untreated GFP; ns: not significant.\u003c/p\u003e","description":"","filename":"ManuscriptFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/a47dccf9efd9ecaf1a241c4e.png"},{"id":87666228,"identity":"95d7174c-41de-4c97-b4f7-6480ffa64dc8","added_by":"auto","created_at":"2025-07-27 11:15:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1134510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eaSyn overexpression upregulated genes of the TGFb pathway in LUHMES cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of the relative gene expression of \u003cem\u003eTGFB1\u003c/em\u003e \u003cstrong\u003e(A)\u003c/strong\u003e, \u003cem\u003eTGFB2\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cem\u003eTGFB3\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e, and \u003cem\u003eTGFBR2\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e were analyzed in untransduced cells (UTCs), aSyn-overexpressing cells, and GFP-expressing cells. \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eTGFB2 \u003c/em\u003ewere found to be up-regulated in aSyn-overexpressing cells only.\u003c/p\u003e\n\u003cp\u003e#: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against UTCs; +++: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 against GFP-expressing cells; ns: not significant.\u003c/p\u003e","description":"","filename":"ManuscriptFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/0523fa4a2ac55fd777aa732e.png"},{"id":101032605,"identity":"01f5cc19-cd28-4864-bebc-9d46a88e88ff","added_by":"auto","created_at":"2026-01-24 08:10:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35734485,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/3d47297c-2a3e-4221-b8f2-17b7e6d1ee60.pdf"},{"id":87666213,"identity":"59f79f1e-835e-493e-9201-737e5dd49260","added_by":"auto","created_at":"2025-07-27 11:15:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1771955,"visible":true,"origin":"","legend":"Reporting summary","description":"","filename":"ajchecklistOscar20250611.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/abd327cfb094fe6ee5388900.pdf"},{"id":87666217,"identity":"dcbfd532-333b-4e4f-8257-6ad5543da5ab","added_by":"auto","created_at":"2025-07-27 11:15:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22478,"visible":true,"origin":"","legend":"\u003cp\u003eFigure legends of the supplementary figures\u003c/p\u003e","description":"","filename":"SupplementaryFile1SuppFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/bef643ccab1c75d74c6de616.docx"},{"id":87666424,"identity":"0f557048-dc12-4f78-b8fb-82b4dc6c81d9","added_by":"auto","created_at":"2025-07-27 11:23:10","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31349686,"visible":true,"origin":"","legend":"\u003cp\u003eFull Western blot images, uncropped\u003c/p\u003e","description":"","filename":"SupplementaryFile2uncroppedWB.docx","url":"https://assets-eu.researchsquare.com/files/rs-6867964/v1/a2e8398120a4973bd481edf4.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Inhibition of TGF-beta signaling protects from alpha-synuclein induced toxicity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is the second-most prevalent neurodegenerative disorder. In PD, the death of dopaminergic neurons in the substantia nigra (SN) leads to a series of observable motor symptoms such as bradykinesia, resting tremor, and rigidity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Histopathologically, PD is defined by the presence of Lewy bodies (LBs) in the brains of PD patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. LBs are intracellular proteinaceous inclusions that mainly contain abnormally aggregated alpha-synuclein (aSyn) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. During the aggregation process of aSyn, oligomeric intermediates between monomers and large insoluble aggregates are formed, and these intermediates are believed to be toxic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We performed a genome-wide siRNA screening in dopaminergic Lund Mesencephalic (LUHMES) cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] with moderate overexpression of wild-type (WT) aSyn [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], in which the knockdown of twelve genes was identified to protect from degeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these, three genes are related to the Transforming Growth Factor-beta (TGFb) canonical pathway, namely Activin A receptor type 1C (\u003cem\u003eACVR1C\u003c/em\u003e, also known as Activin receptor-like kinase 7, \u003cem\u003eALK7\u003c/em\u003e), Dynein light chain roadblock type-1 (\u003cem\u003eDYNLRB1\u003c/em\u003e), and Glycogen synthase kinase 3 beta (\u003cem\u003eGSK3B\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eThe TGFb superfamily is one of the largest signaling pathway families in humans, comprising 33 ligands, 5 type-II receptors, and 7 type-I receptors. Pathways of the TGFb superfamily can be classified according to the ligands, as well as by the downstream cascades. The pathway that involves the phosphorylation of SMAD proteins is known as the canonical pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The binding of TGFb ligands (3 isoforms in humans) to the dimeric TGFb type-II receptor (TGFBR2) activates the TGFb canonical pathway. Upon ligand binding, TGFBR2 recruits and phosphorylates the dimeric TGFb type-I receptor (TGFBR1, also known as activin receptor-like kinase 5, ALK5) [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Together they form a heterotetrameric complex that is internalized into early endosomes, where the heterotetrameric receptor complex can phosphorylate Mothers against decapentaplegic homolog 2 (SMAD2) and/or SMAD3 proteins [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The activated SMAD2/3 complex is then coupled to SMAD4 for nuclear translocation, where the SMAD complex binds its target genes and drives their expression together with a number of different transcription factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ALK7 and DYNLRB1 are closely related to the TGFb pathway, as ALK7 is a type-I receptor that specifically transduces signals from the ligand Nodal [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and DYNLRB1 is involved in the nuclear translocation of phospho-SMAD2 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. GSK3B has been implicated to be involved in the degradation of SMAD3 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. An illustration depicting the TGFb pathway can be found on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the central nervous system, TGFb ligands and receptors are expressed in most parts of the brain, and they are reported to be involved in multiple cellular processes. It has been shown that TGFb ligand treatment could promote the survival and differentiation of primary mesencephalic dopaminergic neurons [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, TGFb signaling has been implicated to play a role in the regulation of neuronal proliferation and apoptosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Due to its large distribution and involvement in multiple cellular processes, several studies looked into whether an increase in TGFb signaling could protect dopaminergic neurons from the toxicity induced by the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP\u003csup\u003e+\u003c/sup\u003e), both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, results from these studies remain contradictory, and the role of TGFb signaling in the context of MPP\u003csup\u003e+\u003c/sup\u003e-induced toxicity remains inconclusive.\u003c/p\u003e\u003cp\u003eIn a previous work, our group discovered that the knockdown of genes involved in the TGFb signaling pathway protected LUHMES cells against aSyn-induced toxicity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hence, in this project, we aimed to investigate the role of the TGFb pathway upon aSyn overexpression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eValidation of the results of the genome-wide siRNA screening\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe validated if the knockdown of \u003cem\u003eALK7\u003c/em\u003e, \u003cem\u003eDYNLRB1\u003c/em\u003e, and \u003cem\u003eGSK3B\u003c/em\u003e could protect cells from aSyn-induced toxicity in our standard cell culture conditions. Utilizing esiRNAs, as employed in the genome-wide screening, we aimed to validate the screening results. In addition, we used siPOOLs siRNAs to further confirm and consolidate our findings. Both types of siRNAs are \u0026lsquo;pools\u0026rsquo; of siRNAs that are produced by enzymatic cleavage of long double-stranded RNA, in which these siRNAs all target the same mRNA. The use of these two distinct siRNA pools helped enhance the reliability of our outcomes.\u003c/p\u003e\u003cp\u003eTo analyze the changes in aSyn-related toxicity, LDH data were normalized relative to the LDH levels measured in aSyn-overexpressing cells. Thus, data from LDH released from aSyn-overexpressing cells are presented as 100%. We first validated the findings of the genome-wide siRNA screening [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] utilizing esiRNAs targeting \u003cem\u003eALK7\u003c/em\u003e, \u003cem\u003eDYNLRB1\u003c/em\u003e, and \u003cem\u003eGSK3B\u003c/em\u003e, which were highlighted in the primary screening. Notably, treatment with esiRNAs against \u003cem\u003eALK7\u003c/em\u003e, \u003cem\u003eDYNLRB1\u003c/em\u003e, and \u003cem\u003eGSK3B\u003c/em\u003e significantly protected LUHMES cells from aSyn-induced cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This reduction was evident in the diminished LDH release as compared to untreated aSyn-overexpressing cells (esiRNA against \u003cem\u003eALK7\u003c/em\u003e: 68.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; esiRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e: 72.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; esiRNA against \u003cem\u003eGSK3B\u003c/em\u003e: 76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; each compared to untransfected aSyn-overexpressing cells; 100\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%). By using the siPOOL siRNAs, we also confirmed that the transfection with siPOOL siRNAs against \u003cem\u003eDYNLRB1\u003c/em\u003e and \u003cem\u003eGSK3B\u003c/em\u003e protected aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), as quantified by the reduction in LDH released from aSyn-overexpressing cells (siPOOL siRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e: 74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; siPOOL siRNA against \u003cem\u003eGSK3B\u003c/em\u003e: 87.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; each compared to untransfected aSyn-overexpressing cells; 100\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%). However, in contrast to the esiRNA, the treatment with the siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e did not protect aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether the protection against aSyn-induced toxicity was the consequence of a specific knockdown of the respective target genes, or an off-target effect, we investigated the expression of GSK3B, DYNLRB1, and ALK7 in untransduced cells and aSyn-overexpressing cells after the transfection with the siRNAs. By performing WB analyses, we confirmed that the esiRNA against \u003cem\u003eGSK3B\u003c/em\u003e led to a significant down-regulation of GSK3B protein in both untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; full-size Western blot in Supplementary File 2 Fig. A). Similarly, the siPOOL siRNA against \u003cem\u003eGSK3B\u003c/em\u003e markedly reduced the expression of GSK3B protein in both untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; full-size Western blot in Supplementary File 2 Fig. B). To reinforce our hypothesis that reducing GSK3B levels protects LUHMES cells from aSyn-induced toxicity, we tested treated LUHMES cells with two different inhibitors of GSK3B, tideglusib [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and GSK3B inhibitor-VIII (GSK8) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Both tideglusib and GSK8 led to a significant reduction in LDH release in aSyn-overexpressing cells, demonstrating their protective efficacy. Tideglusib reduced aSyn-induced toxicity at concentrations of 10 \u0026micro;M (88.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 20 \u0026micro;M (72.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; untreated aSyn-overexpressing cells: 100\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03%; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Similarly, GSK8 treatment resulted in a significant reduction of LDH release in aSyn-overexpressing cells (100\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%) at concentrations of 1 \u0026micro;M (84.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 2 \u0026micro;M (57.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Additionally, aSyn overexpression significantly elevated GSK3B levels as compared to untransduced cells, highlighting the role of GSK3B in aSyn-induced toxicity in our cell model. Collectively, our results suggest that reducing GSK3B availability, either through the knockdown of GSK3B or by direct pharmacological inhibition, protects LUHMES cells from aSyn-induced toxicity.\u003c/p\u003e\u003cp\u003eDue to the lack of commercially available WB antibodies for DYNLRB1, we assessed the knockdown efficacy of both the esiRNAs and the siPOOL siRNAs against \u003cem\u003eDYNLRB1\u003c/em\u003e by qPCR. The esiRNA against \u003cem\u003eDYNLRB1\u003c/em\u003e significantly reduced \u003cem\u003eDYNLRB1\u003c/em\u003e mRNA expression in both untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). We also identified a significant up-regulation of \u003cem\u003eDYNLRB1\u003c/em\u003e associated with aSyn overexpression. Similarly, the siPOOL siRNA targeting \u003cem\u003eDYNLRB1\u003c/em\u003e led to a significant reduction in \u003cem\u003eDYNLRB1\u003c/em\u003e mRNA expression in untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), indicating that both siRNAs against \u003cem\u003eDYNLRB1\u003c/em\u003e were effective.\u003c/p\u003e\u003cp\u003eSince transfection with the siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e did not protect against aSyn-induced toxicity, we conducted a deeper analysis of \u003cem\u003eALK7\u003c/em\u003e expression in LUHMES cells. In contrast to the gene expression of the other two type-I receptors of the TGFb pathway (\u003cem\u003eALK4\u003c/em\u003e and \u003cem\u003eALK5\u003c/em\u003e), as well as the housekeeping genes (\u003cem\u003ehuUBQLN1\u003c/em\u003e and \u003cem\u003ePOLR2A\u003c/em\u003e), \u003cem\u003eALK7\u003c/em\u003e mRNA was only detectable after 34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cycles, indicating extremely low expression levels in LUHMES cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Subsequently, we examined whether the esiRNA and the siPOOL siRNA targeting \u003cem\u003eALK7\u003c/em\u003e could reduce \u003cem\u003eALK7\u003c/em\u003e expression.\u003c/p\u003e\u003cp\u003eThe esiRNA against \u003cem\u003eALK7\u003c/em\u003e significantly reduced \u003cem\u003eALK7\u003c/em\u003e expression only in aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As the treatment with the esiRNA, but not the siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e, conferred protection to LUHMES cells against aSyn-induced toxicity, we hypothesized that the protectiveness observed with the esiRNA against \u003cem\u003eALK7\u003c/em\u003e might arise from off-target activities. Considering the close phylogenetic similarity between ALK7 and the TGFb pathway receptors ALK4 and ALK5 [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], we hypothesized that \u003cem\u003eALK4\u003c/em\u003e and \u003cem\u003eALK5\u003c/em\u003e might be potential off-targets of the \u003cem\u003eALK7\u003c/em\u003e esiRNA. However, our analysis revealed that the esiRNA against \u003cem\u003eALK7\u003c/em\u003e did not down-regulate \u003cem\u003eALK4\u003c/em\u003e mRNA levels in either untransduced cells or aSyn-overexpressing cells, suggesting that \u003cem\u003eALK4\u003c/em\u003e was not the off-target of the \u003cem\u003eALK7\u003c/em\u003e esiRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the esiRNA against \u003cem\u003eALK7\u003c/em\u003e significantly reduced \u003cem\u003eALK5\u003c/em\u003e mRNA expression in aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating that \u003cem\u003eALK5\u003c/em\u003e was indeed an off-target.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e also resulted in a reduction in \u003cem\u003eALK7\u003c/em\u003e expression only in aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, unlike the esiRNA, the siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e did not lead to significant changes in the expression of both \u003cem\u003eALK4\u003c/em\u003e and \u003cem\u003eALK5\u003c/em\u003e in either untransduced cells or aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Given that the protection against aSyn-induced toxicity was only observed after the transfection of esiRNA against \u003cem\u003eALK7\u003c/em\u003e, for which we identified \u003cem\u003eALK5\u003c/em\u003e as off-target, together with the fact that the expression of \u003cem\u003eALK7\u003c/em\u003e was very low in LUHMES cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), we proposed that \u003cem\u003eALK5\u003c/em\u003e knockdown, rather than \u003cem\u003eALK7\u003c/em\u003e knockdown, was responsible for the observed protective effect against aSyn-induced toxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eALK5 and its downstream pathway protected LUHMES cells from aSyn-induced toxicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify that the knockdown of \u003cem\u003eALK5\u003c/em\u003e protected against aSyn-induced toxicity, we first validated if the siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e could effectively and specifically reduce \u003cem\u003eALK5\u003c/em\u003e mRNA expression. The siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e significantly reduced \u003cem\u003eALK5\u003c/em\u003e expression in both untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). We also confirmed that the siPOOL siRNA against \u003cem\u003eALK5\u003c/em\u003e did not lead to changes in the mRNA expression of \u003cem\u003eALK4\u003c/em\u003e or \u003cem\u003eALK7\u003c/em\u003e in both untransduced cells and aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). We then confirmed that the knockdown of \u003cem\u003eALK5\u003c/em\u003e could protect LUHMES cells from aSyn-induced toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The knockdown of \u003cem\u003eALK5\u003c/em\u003e protected LUHMES cells from aSyn-induced toxicity, as quantified by a reduction of LDH release (from 100\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0% as in untreated aSyn-overexpressing cells to 87.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The protective effect of \u003cem\u003eALK5\u003c/em\u003e knockdown was specific against aSyn-induced toxicity, since the knockdown of \u003cem\u003eALK5\u003c/em\u003e did not lead to changes in cell viability in either untransduced cells or GFP-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we used an inhibitor of ALK5, SM16 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], to further confirm if the inhibition of ALK5 protected against aSyn-induced toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As anticipated, SM16 treatment led to a significant dose-dependent protection against aSyn-induced toxicity. In particular, SM16 treatment led to a reduction of LDH release in aSyn-overexpressing cells from 100\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2% to 77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2% at 10 \u0026micro;M (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), to 68.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8% at 20 \u0026micro;M (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and to 55.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8% at 40 \u0026micro;M (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar to the observation made after the knockdown of \u003cem\u003eALK5\u003c/em\u003e, the protection by the treatment with SM16 was specific to aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWe then explored if the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e and/or \u003cem\u003eSMAD3\u003c/em\u003e, the downstream targets of ALK5, could confer protection against aSyn-induced toxicity. Interestingly, only the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e, but not \u003cem\u003eSMAD3\u003c/em\u003e, demonstrated a protective effect. When compared to the LDH released from untreated aSyn-overexpressing cells, the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e led to a reduction of LDH release from 100\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4% to 88.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the protection after the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e was only observed in aSyn-overexpressing cells, indicating an aSyn-dependent effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Due to the high sequence homology between \u003cem\u003eSMAD2\u003c/em\u003e and \u003cem\u003eSMAD3\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we verified the specificities of siPOOL siRNAs for their respective targets. In both untransduced cells and aSyn-overexpressing cells, we found that the siPOOL siRNA against \u003cem\u003eSMAD2\u003c/em\u003e specifically reduced \u003cem\u003eSMAD2\u003c/em\u003e expression without affecting the expression of \u003cem\u003eSMAD3\u003c/em\u003e (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In the same manner, the siPOOL siRNA against \u003cem\u003eSMAD3\u003c/em\u003e specifically reduced \u003cem\u003eSMAD3\u003c/em\u003e expression without affecting the expression of \u003cem\u003eSMAD2\u003c/em\u003e (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Interestingly, the expression of \u003cem\u003eSMAD2\u003c/em\u003e was much higher than \u003cem\u003eSMAD3\u003c/em\u003e (approximately 10 threshold cycles higher, i.e. over two orders of magnitude) in LUHMES cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSince the TGFb pathway is involved in the differentiation of dopaminergic neurons [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], we performed a control experiment to verify that the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e did not interfere with the differentiation of LUHMES cells. We confirmed that the mRNA expression of tyrosine hydroxylase (\u003cem\u003eTH\u003c/em\u003e), a dopaminergic neuronal marker, was not significantly altered by the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e in untransduced cells, aSyn-overexpressing cells, and GFP-expressing cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). To further ensure that the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e did not impact on the transcriptional activities of the adenoviral vectors used in our experiments, we measured the expression of aSyn on both transcript and protein levels. The knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e did not affect the expression of \u003cem\u003eSNCA\u003c/em\u003e, the gene that encodes aSyn, nor the level of aSyn protein (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG, H; full-size Western blot in Supplementary File 2 Fig. C) in untransduced cells, aSyn-overexpressing cells, or GFP-expressing cells. Concurrently, the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, \u003cem\u003eSMAD3\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e did not interfere with the expression of GFP in GFP-expressing cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI). These data emphasize that the protection against aSyn-induced toxicity resulting from the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e was not caused by an interference with differentiation of LUHMES cells, or by a change in the transcriptional activities of the adenoviral vectors. In addition, as \u003cem\u003eSMAD3\u003c/em\u003e expression was low in LUHMES cells and its knockdown did not yield observable phenotypic changes such as protection against aSyn-induced toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) or changes in the expression of aSyn (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG, H), we excluded \u003cem\u003eSMAD3\u003c/em\u003e in the following experiments.\u003c/p\u003e\u003cp\u003eWe then performed immunocytochemistry to investigate whether the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e also ameliorated loss of neuronal network density (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; untransduced cells: Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA; GFP-expressing cells: Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). We quantified the density of neuronal network by counting the number of quadruple points per cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Each quadruple point reflects a dendritic junction that contains or connects four distinct neurite branches, serving as an indicator of neuronal network complexity. In untransduced cells and GFP-expressing cells, the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, or \u003cem\u003eDYNLRB1\u003c/em\u003e did not significantly alter neuronal network density, as compared to respective control cells without siRNA transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). As expected, aSyn overexpression substantially decreased the density of the neuronal network, as quantified by a reduction in the number of quadruple points per cell in comparison to the untransduced cells. (untransduced cells: 199.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7; aSyn-overexpressing cells: 84.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Such reduction of the neuronal network complexity in aSyn-overexpressing cells was counteracted by the knockdown of \u003cem\u003eALK5\u003c/em\u003e and \u003cem\u003eSMAD2\u003c/em\u003e, as we observed a significant increase in the number of quadruple points per cell in comparison to the untransfected aSyn-overexpressing cells (\u003cem\u003eALK5\u003c/em\u003e knockdown: 154.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eSMAD2\u003c/em\u003e knockdown: 150.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). On the other hand, the knockdown of \u003cem\u003eDYNLRB1\u003c/em\u003e also led to an increase in the density of neuronal network of aSyn-overexpressing cells, however it did not reach statistical significance (126.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.2; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). To further support these findings, we also detected activated caspases 3/7, an apoptotic marker, using a fluorescent dye (CellEvent\u0026trade;; Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC, D). In aSyn-overexpressing cells, the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e led to an obvious reduction in the expression of caspase 3/7 (from 100\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% to 65.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.73%).\u003c/p\u003e\u003cp\u003eIn summary, we demonstrated that reducing TGFb signaling, specifically via the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e, protected LUHMES cells against aSyn-induced toxicity. In particular, the knockdown of ALK5 prevented the loss of neuronal network density, whereas the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e not only preserved neuronal network density but also inhibited apoptotic activation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe over-activation of TGFb pathway is detrimental to aSyn-overexpressing cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince the inhibition of TGFb signaling protected against aSyn-induced toxicity, we explored whether activating this pathway would have the opposite effect. Therefore, we treated aSyn-overexpressing cells with recombinant human TGFb proteins. We used TGFb ligands at 10 ng/mL, a dose that was previously reported to effectively activate the TGFb pathway [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In comparison to untreated aSyn-overexpressing cells (indicated as 100%), treatment with TGFb1, TGFb2, and TGFb3 led to an increase of LDH release in aSyn-overexpressing cells (TGFb1: 113.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TGFb2: 114.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TGFb3: 112.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Furthermore, the increase in cytotoxicity induced by TGFb ligand treatment was also reflected by a decrease in neuronal network density. Specifically, following the treatment with TGFb1, we observed a significant decrease in the number of quadruple points per cell, dropping from 109.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 in untreated aSyn-overexpressing cells, to 72.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Notably, this cytotoxic effect caused by TGFb ligand treatment was exclusive to aSyn-overexpressing cells. In contrast, untransduced or GFP-expressing cells treated with TGFb ligand (10 ng/mL) exhibited no significant changes in cell viability (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, B) or neuronal network density (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD, E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo gain further evidence for the role of TGFb signaling in aSyn-induced toxicity, we examined if the increase in cytotoxicity after TGFb treatment could be mitigated by knocking down key genes in the TGFb signaling pathway. Therefore, aSyn-overexpressing LUHMES cells were treated with TGFb1, TGFb2, or TGFb3 and transfected with siPOOL siRNAs against \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e. As expected, the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e effectively ameliorated the increase in toxicity resulted from the treatment with TGFb ligands. In TGFb1-treated aSyn-overexpressing cells, a reduction in LDH release was observed upon the knockdown of \u003cem\u003eALK5\u003c/em\u003e (67.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eSMAD2\u003c/em\u003e (86.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eDYNLRB1\u003c/em\u003e (86.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), in comparison to the LDH released from TGFb1-treated aSyn-overexpressing cells (113.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0%; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similarly, in TGFb2-treated aSyn-overexpressing cells, a reduction in LDH release was observed upon the knockdown of \u003cem\u003eALK5\u003c/em\u003e (67.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eSMAD2\u003c/em\u003e (89.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eDYNLRB1\u003c/em\u003e (86.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), in comparison to the LDH released from TGFb2-treated aSyn-overexpressing cells (114.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The same trend was also observed in TGFb3-treated aSyn-overexpressing cells, where a reduction in LDH release was observed upon the knockdown of \u003cem\u003eALK5\u003c/em\u003e (67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eSMAD2\u003c/em\u003e (89.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and \u003cem\u003eDYNLRB1\u003c/em\u003e (86.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), in comparison to TGFb3-treated aSyn-overexpressing cells (112.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The decrease in cytotoxicity resulted from the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e was also reflected in the increase in the density of neuronal network of TGFb1-treated aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Specifically, the knockdown of \u003cem\u003eALK5\u003c/em\u003e in TGFb1-treated aSyn-overexpressing cells led to a significant increase in the number of quadruple points per cell from 72.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 to 147.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e to 161.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the knockdown of \u003cem\u003eDYNLRB1\u003c/em\u003e to 111.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02). In summary, with these experiments, we demonstrated that an increase in TGFb signaling via treatment with TGFb ligands exacerbates toxicity in aSyn-overexpressing cells, and this detrimental effect can be mitigated through the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn addition, we also performed a staining of activated caspases 3/7 as apoptotic marker to complement our findings (Supplementary Fig. S4A, B). Even though TGFb1 treatment in aSyn-overexpressing cells did not lead to a significant increase in the intensity of caspase 3/7 signals as compared to untreated aSyn-overexpressing cells (100\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3%), a significant decrease in caspase 3/7 signals was observed upon the knockdown of \u003cem\u003eALK5\u003c/em\u003e (65.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%), \u003cem\u003eSMAD2\u003c/em\u003e (57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8%), or \u003cem\u003eDYNLRB1\u003c/em\u003e (77.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%), indicating a significant decrease in the number of apoptotic cells. These results further illustrate that the knockdown of TGFb signaling can effectively counteract the increase of aSyn-induced toxicity resulting from TGFb1 treatment.\u003c/p\u003e\u003cp\u003eTo further understand why TGFb ligand treatment exacerbated aSyn-induced toxicity, we examined if TGFb ligand treatment influenced \u003cem\u003eSNCA\u003c/em\u003e expression. Interestingly, we observed that TGFb ligand treatment led to an additional up-regulation of the \u003cem\u003eSNCA\u003c/em\u003e mRNA in aSyn-overexpressing cells (untreated aSyn-overexpressing cells: 24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0; TGFb1 treatment: 29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07; TGFb2 treatment: 33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TGFb3 treatment: 34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, this treatment did not change the expression of the \u003cem\u003eSNCA\u003c/em\u003e in untransduced cells or GFP-expressing cells. To determine if TGFb ligand treatment had an influence on the transcriptional activities of the adenoviral vectors, we then investigated if TGFb ligand treatment would also lead to an up-regulation of GFP in GFP-expressing cells. However, there was no significant increase in the expression of the GFP transcript following treatment with TGFb1, TGFb2, or TGFb3 in GFP-expressing cells (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). These findings indicate that the up-regulation of \u003cem\u003eSNCA\u003c/em\u003e induced by TGFb ligand treatment was specific to the aSyn-overexpressing condition and not due to an increase of the transcriptional activities of the adenoviral vectors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscripts of the TGFb pathway were up-regulated upon aSyn-overexpression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we examined if the overexpression of aSyn led to changes in the expression of genes involved in the TGFb pathway. We have previously identified a significant increase in the expression of \u003cem\u003eSMAD2\u003c/em\u003e (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) and \u003cem\u003eSMAD3\u003c/em\u003e (4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD) in aSyn-overexpressing cells. In contrast, we did not observe an up-regulation of \u003cem\u003eALK4\u003c/em\u003e or \u003cem\u003eALK5\u003c/em\u003e in aSyn-overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To expand upon these findings, we analyzed the expression levels of four additional key TGFb pathway genes, namely \u003cem\u003eTGFB1\u003c/em\u003e, \u003cem\u003eTGFB2\u003c/em\u003e, \u003cem\u003eTGFB3\u003c/em\u003e, and \u003cem\u003eTGFBR2\u003c/em\u003e in untransduced cells, aSyn-overexpressing cells, and GFP-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We found that aSyn-overexpressing cells, in comparison to untransduced cells, showed higher expression levels of \u003cem\u003eTGFB1\u003c/em\u003e (1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and \u003cem\u003eTGFB2\u003c/em\u003e (3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, the expressions of \u003cem\u003eTGFB1\u003c/em\u003e, \u003cem\u003eTGFB2\u003c/em\u003e, \u003cem\u003eTGFB3\u003c/em\u003e, and \u003cem\u003eTGFBR2\u003c/em\u003e in untransduced LUHMES cells were comparable (between 28th to 32nd threshold cycle, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF). On the other hand, we did not observe any differences in the expression of genes of the TGFb pathway between untransduced cells and GFP-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG). This suggests that TGFb signaling was up-regulated because of aSyn overexpression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreviously, in a genome-wide siRNA screening of a PD cell model, we identified that the knockdown of three genes (\u003cem\u003eALK7\u003c/em\u003e, \u003cem\u003eDYNLRB1\u003c/em\u003e, and \u003cem\u003eGSK3B\u003c/em\u003e), which are associated with the TGFb signaling pathway, was found to mitigate aSyn-induced toxicity (H\u0026ouml;llerhage et al., 2025). Hence, in this project, we investigated the role of the TGFb signaling pathway in aSyn-induced toxicity. Consistent with our initial findings in the siRNA screening, the knockdown of \u003cem\u003eDYNLRB1\u003c/em\u003e and \u003cem\u003eGSK3B\u003c/em\u003e by different knockdown paradigms (i.e. esiRNA and siPOOL siRNA) protected human dopaminergic neuron-like cells (LUHMES cells) from aSyn-induced toxicity. In addition, the treatment with two inhibitors of GSK3B, tideglusib and GSK8, also demonstrated a protective effect. On the other hand, while we identified that the transfection of the esiRNA provided protection against aSyn-induced toxicity, the siPOOL siRNA against \u003cem\u003eALK7\u003c/em\u003e did not. A deeper analysis revealed that the actual target of the \u003cem\u003eALK7\u003c/em\u003e esiRNA was \u003cem\u003eALK5\u003c/em\u003e, the gene coding for type-I receptor of the TGFb pathway. We subsequently proved that the knockdown of \u003cem\u003eALK5\u003c/em\u003e safeguarded LUHMES cells from aSyn-induced toxicity. Furthermore, treatment with a specific ALK5 inhibitor (SM16) also confered a similar protective effect. In addition to \u003cem\u003eALK5\u003c/em\u003e, we identified that the knockdown of \u003cem\u003eSMAD2\u003c/em\u003e, a downstream target of ALK5, also protected against aSyn-induced toxicity.\u003c/p\u003e\u003cp\u003eIn addition to this demonstration that the reduction of TGFb signaling was protective against aSyn-induced toxicity, we found that the opposite (i.e. stimulation of TGFb signaling by ligand treatment) was detrimental in our PD cell model. Furthermore, the knockdown of TGFb-associated genes abolished the adverse effects evoked by TGFb ligands. Moreover, we found that TGFb ligand treatment led to an increase in \u003cem\u003eSNCA\u003c/em\u003e expression in our PD cell model, and aSyn-overexpression led to an up-regulation of \u003cem\u003eTGFB1\u003c/em\u003e, \u003cem\u003eTGFB2\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eSMAD3\u003c/em\u003e in LUHMES cells.\u003c/p\u003e\u003cp\u003eIn this project, we have provided compelling evidence that the reduction of TGFb signaling protected dopaminergic neurons from aSyn-induced toxicity, and an increase in TGFb signaling led to an elevation of aSyn-induced toxicity. Even though TGFb ligands were reported to be generally neurotrophic to dopaminergic neurons [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], few studies have explored the therapeutic potential of the TGFb signaling pathway in PD. In these toxin-based PD models, mouse or rat dopaminergic primary neurons or mice were treated with a mitochondrial complex I inhibitor MPP\u003csup\u003e+\u003c/sup\u003e or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; precursor of MPP\u003csup\u003e+\u003c/sup\u003e) that specifically kills dopaminergic neurons [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the outcomes varied. On one hand, one study reported that dopaminergic neurons in rat mesencephalic primary cultures that had been treated with TGFb1 were less susceptible to MPP\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In line with that, another study showed that an increase in TGFb signaling via the expression of a constitutively active ALK5 protected mouse dopaminergic neurons in the SN from MPTP-induced toxicity \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. On the other hand, one study reported that the adenoviral overexpression of TGFb1 in the striatum led to a decrease in the number of midbrain dopaminergic neurons in MPTP-injected mice \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These conflicting outcomes highlight the uncertainty surrounding the role of TGFb signaling in toxin-based PD models. In distinction from previous studies, we investigated the role of TGFb signaling in a human PD cell model that shows toxicity induced by human aSyn. One advantage of our cell model is its close resemblance to neurons that degenerate in PD, providing a more accurate representation of the disease in humans. One study demonstrated that aSyn-toxicity models could replicate human PD pathology more closely than toxin models like 6-hydroxydopamine (6-OHDA) \u003cem\u003ein vivo\u003c/em\u003e. The overexpression of aSyn resulted in a progressive loss of dopaminergic neurons that occurred in the course of eight weeks, accompanied by axonal swellings and an impairment in post-synaptic activity. These features were absent in 6-OHDA models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our human aSyn toxicity cell model enabled us to investigate how changes in the TGFb signaling led to changes in aSyn-induced cytotoxicity, as well as to observe how aSyn and the genes/proteins of the TGFb pathway could modulate the expression of each other. Importantly, we found that TGFb ligand treatment induced an up-regulation of the \u003cem\u003eSNCA\u003c/em\u003e gene upon aSyn overexpression. Similar contradictions in neuroprotection were observed with glial cell-derived neurotrophic factor (GDNF), another member of the TGFb ligand family. While GDNF treatment was protective against MPTP-induced toxicity in mice and monkey \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], it failed to protect dopaminergic neurons against aSyn-induced toxicity in rats \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, a recent clinical trial in which GDNF was infused into the putamen of PD patients via a skull-mounted transcutaneous port and four separate intraputamental infusion catheters also failed to meet its primary endpoint, as GDNF treatment did not lead to significant improvement of the motor symptoms in comparison to placebo control [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These results underscore the critical importance of selecting appropriate experimental models, as differences in experimental models could result in entirely opposite conclusions.\u003c/p\u003e\u003cp\u003eThis study employed three distinct measurements to assess cell viability/cell death to verify whether the inhibition of TGFb signaling in aSyn-overexpressing cells could provide protection against aSyn-induced toxicity. Results from the LDH release assay and the network analysis indicated that suppressing TGFb signaling significantly reduced cell death in aSyn overexpressing cells and simultaneously led to a significant increase in the density of neuronal network. Furthermore, staining for activated caspases 3/7 confirmed the protective effect of reducing TGFb signaling. In TGFb1-treated aSyn-overexpressing cells, the knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eDYNLRB1\u003c/em\u003e all led to a significant reduction in the caspase signals. Even though all three assays assess cell death or cell viability, they differ fundamentally. LDH assay measures LDH released into the culture medium, which reflects membrane integrity; the network analysis is an indicator for neuronal network complexity; and the staining for activated caspases 3/7 serves as an apoptotic marker. These methodological differences between the assays could account for the differences in results. Nonetheless, our findings highlight SMAD2 can be a promising therapeutic against PD, since knockdown of \u003cem\u003eSMAD2\u003c/em\u003e showed pronounced protective efficacy consistently across all three readout methods.\u003c/p\u003e\u003cp\u003eIn this project, we identified an up-regulation of \u003cem\u003eTGFB1\u003c/em\u003e, \u003cem\u003eTGFB2\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, and \u003cem\u003eSMAD3\u003c/em\u003e as consequence of aSyn-overexpression in LUHMES cells. In accordance with this, it has been previously shown that TGFb1 is upregulated in the striatum of \u003cem\u003epost mortem\u003c/em\u003e PD patients [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, it has been reported previously that TGFb2 was elevated in the vCSF of PD patients in comparison to healthy controls [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The up-regulation of genes and proteins of the TGFb pathway in multiple regions of the brain could indicate a global up-regulation in the brains of PD patients. However, the expression patterns of the genes and proteins of the TGFb pathway in the brains of PD patients are not well understood. In particular, it is unknown whether such up-regulation follows the spread of aSyn pathology. Furthermore, the time point at which such an up-regulation could occur is unknown. To understand if the up-regulation of genes and proteins of the TGFb pathway in the brains of PD patients is global and progressive, deeper analyses of various brain regions would be needed, which was beyond the scope of this study.\u003c/p\u003e\u003cp\u003eOur results offer valuable insights for the development of potential drugs against PD. As an example, we identified two inhibitors of GSK3B and one inhibitor of ALK5, which significantly protected dopaminergic neurons against aSyn-induced toxicity. Among them, tideglusib has already been used in clinical trials in patients with progressive supranuclear palsy [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], meaning existing data on its dosage and safety are already available. In the context of PD, tideglusib treatment protected dopaminergic neurons in mice against MPP\u003csup\u003e+\u003c/sup\u003e-induced toxicity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Together with our findings that tideglusib protected dopaminergic neurons from aSyn-induced toxicity, it would be worthwhile to continue with clinical trials of tideglusib in PD. On the other hand, SM16 has been used \u003cem\u003ein vivo\u003c/em\u003e in rodents to inhibit tumor growth, and therefore its safety \u003cem\u003ein vivo\u003c/em\u003e has also been proven [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, SM16 has never been used in clinical trials or in experiments that involve non-human primates \u003cem\u003ein vivo\u003c/em\u003e. As our results provided clear indications that the use of SM16 strongly protected dopaminergic neurons from aSyn-induced toxicity \u003cem\u003ein vitro\u003c/em\u003e, it would be worthwhile to assess if SM16 also exerts its protectiveness against aSyn-induced toxicity \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, our findings highlight the detrimental role of TGFb signaling in relation to aSyn-induced toxicity. Furthermore, we observed that the reduction of TGFb signaling protected against aSyn-induced toxicity. Our results also suggest that \u003cem\u003eSNCA\u003c/em\u003e expression and TGFb pathway gene expression may influence each other. The alignment between our \u003cem\u003ein vitro\u003c/em\u003e results and the published observations in human \u003cem\u003epost mortem\u003c/em\u003e brains as well as vCSF of PD patients [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] support our findings and thus could be the basis for the development of TGFb inhibiting drugs as therapeutic approach for PD and related synucleinopathies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eCell Culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLUHMES cells (CRL-2927, American Type Culture Collection), were used in all \u003cem\u003ein vitro\u003c/em\u003e experiments. Cells were kept at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and moisturized air. The cell culture flasks (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were pre-coated at 37\u0026deg;C overnight with 0.1 mg/mL poly-L-ornithine (PLO; Sigma-Aldrich, St. Louis, MO, USA). For proliferation, LUHMES cells were maintained in proliferation medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, DMEM/F12 (Sigma-Aldrich) with 1% N2 supplement (Thermo Fisher Scientific), and 0.04 \u0026micro;g/mL fibroblast growth factor-basic, PeproTech, Rocky Hill, CT, USA). For the actual experiments, multi-well plates or flasks (Thermo Fisher Scientific) were pre-coated with 0.1 mg/mL PLO (37\u0026deg;C, overnight) and subsequently with 5 \u0026micro;g/mL fibronectin (FN; R\u0026amp;D Systems, Minneapolis, MN, USA) at 37\u0026deg;C overnight. To initiate the differentiation process, differentiation medium consistent of DMEM/F12 (Sigma-Aldrich) with 1% N2 supplement (Thermo Fisher Scientific), 1 \u0026micro;g/mL tetracycline (Sigma-Aldrich), 0.49 mg/mL dibutyryl-cyclic adenosine monophosphate (Sigma-Aldrich), and 2 ng/\u0026micro;l glial cell line-derived neurotrophic factor (R\u0026amp;D Systems) was used [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Prior the coating with FN, multi-well plates or flasks were washed three times with sterile water to remove excess PLO. Before seeding, the multi-well plates or flasks were washed three times with sterile phosphate buffered saline (PBS; Sigma-Aldrich) to remove excess FN. Cell cultures are routinely tested for mycoplasma to ensure they are not contaminated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdenoviral transduction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOn days \u003cem\u003ein vitro\u003c/em\u003e (DIV) 2, cells were transduced with adenoviral vectors (Charles Rivers Laboratories, Leiden, the Netherlands) to overexpress human wild-type aSyn or GFP under a cytomegalovirus promotor. 24 hours (h) post-transduction, cells were washed three times with Hank\u0026rsquo;s Balanced Salt Solution (HBSS; Sigma-Aldrich) to remove residual virus and were then replenished with fresh DM, as previously described [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003esiRNA transfection / ligand \u0026amp; inhibitor treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApproximately 4\u0026ndash;6 h after the removal of viral vectors, cells were transfected with siRNAs and/or were treated with ligands or inhibitors. Two different types of siRNAs were used, namely MISSION\u0026reg; endoribonuclease-prepared siRNA (esiRNA, Sigma-Aldrich) and siPOOLs siRNAs (siTOOLs, Martinsried, Germany). Before transfection, the siRNAs were mixed with Opti-MEM\u0026trade; medium (Thermo Fisher Scientific) and the transfection reagent Lipofectamine\u0026trade; RNAiMax (Thermo Fisher Scientific). The mixture was then vortexed and incubated for 20 minutes (min) prior to treating the cells. The final concentrations of the esiRNA were 200 ng/\u0026micro;L and those of siPOOL were 5 nM. Ligands or inhibitors were diluted in DM into their respective final concentrations immediately before treating the cells.\u003c/p\u003e\u003cp\u003eThe following ligands and inhibitors were used: Recombinant human TGFb1 protein (R\u0026amp;D Systems), recombinant human TGFb2 protein (R\u0026amp;D Systems), recombinant human TGFb3 protein (R\u0026amp;D Systems), tideglusib (Sigma-Aldrich), GSK3B-inhibitor VIII (Sigma-Aldrich), or SM16 (Tocris, Minneapolis, MN, USA)\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactate dehydrogenase cytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLactate dehydrogenase (LDH) as measure for cytotoxicity was quantified by spectrophotometric measurement of the absorbance at 340 nm, which is an indirect measurement of the consumption of nicotinamide adenine dinucleotide (NADH) by LDH. Briefly, culture medium was harvested from cells on DIV8, and was mixed with a reaction buffer consistent of 74.24 mM Tris/HCl (Sigma-Aldrich), 185.6 mM NaCl (Sigma-Aldrich), 3.2 mM pyruvate (Sigma-Aldrich), and 4 mM NADH (Sigma-Aldrich) in water. We then measured the absorbance of the mixture at 340 nm and 420 nm with the use of a plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). Medium from cells lysed with 10% Triton X-100 (Sigma-Aldrich) 1 h before the assay was used as positive control. Unless otherwise specified, all LDH data were normalized to the LDH measured in aSyn-overexpressing cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal-time quantitative polymerase chain reaction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe used RNeasy\u0026reg; Mini kit (Qiagen, Hilden, Germany) for the extraction of RNA. Briefly, we harvested cells on DIV6 using RLT buffer (activated with 1% beta-mercaptoethanol; Sigma-Aldrich) and cell scrapers. Then, the samples were run through genomic DNA eliminator columns. The resultant flow-through was mixed thoroughly with 70% ethanol, and the mixture was eluded through RNA spin-columns. RNase-free water was used to dissolve the RNA extracted in the RNA spin-columns. RNA concentration was determined using a NanoDrop\u0026trade; 2000 Microvolume Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the iScript\u0026trade; cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). A CFX96 Touch\u0026trade; Real-Time PCR Detection System (Bio-Rad Laboratories) was used for the real-time quantitative polymerase chain reaction (qPCR) and SYBR\u0026trade; Select Master Mix for CFX (Thermo Fisher Scientific) was used as a dye to indicate double-stranded DNA formed in the qPCR process.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were harvested on DIV6 using M-PER\u0026trade; lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). The lysates were then centrifuged at 4\u0026deg;C, 10 min, 13,000 \u003cem\u003eg\u003c/em\u003e to remove cell debris. The concentration of each lysate was then determined by performing the bicinchoninic acid (BCA) assay using the Pierce\u0026trade; BCA protein assay kit (Thermo Fisher Scientific). Then, protein samples were loaded on 4\u0026ndash;12% Criterion\u0026trade; XT bis-tris precast protein gels (Bio-Rad Laboratories) with 2-(N-morpholino)ethanesulfonic acid (MES) running buffer. After the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the gels were transferred onto pre-assembled 0.2 \u0026micro;m polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories) in a Trans-Blot\u0026reg; SD semi-dry blotter (Bio-Rad Laboratories). After the transfer, membranes were incubated with 0.4% paraformaldehyde (PFA; Sigma-Aldrich) for 30 min. The membranes were then rinsed with PBS three times (5 min each), followed by an incubation in blocking solution (30% Roti\u0026reg;Block (Carl Roth, Karlsruhe, Germany)) in Tris-Buffered Saline with 0.1% Tween 20 (TBST; Sigma-Aldrich) for 1 h at room temperature (RT). Then, the membranes were incubated with primary antibodies diluted with 10% Roti\u0026reg;Block (Carl Roth)/TBST solution at 4\u0026deg;C overnight. On the next day, membranes were rinsed three times with TBST (5 min each) to remove the primary antibodies and then were incubated with the secondary antibodies (diluted in TBST) for 2 h at RT. After the incubation with the secondary antibodies, the membranes were rinsed three times with TBST (5 min each) to remove the secondary antibodies. Then, the membranes were incubated with Clarity\u0026trade; Western ECL Substrate (Bio-Rad Laboratories). An Odyssey\u0026reg; Fc Imager (LI-COR Biotechnology, Lincoln, NE, USA) was used to obtain images of the membranes.\u003c/p\u003e\u003cp\u003eThe following primary antibodies were used: rabbit anti-aSyn (1:500; 701085, Thermo Fisher Scientific); rabbit anti-GAPDH (1:1000; 2118, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-GSK3B (1:5000; ab32391, Abcam, Cambridge, UK). The following secondary antibodies were used: HRP-coupled goat anti-mouse IgG (1:5000; PI-2000, Vector Laboratories, Burlingame, CA, USA); HRP-coupled goat anti-rabbit IgG (1:5000; PI-1000, Vector Laboratories).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunocytochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrior the start of experiments, glass cover slips (Bellco Glass Inc., Vineland, NJ, USA) were coated in 24-well plates (Thermo Fisher Scientific) with PLO and FN before seeding the cells. The coating procedures and the cell culture were performed as described above. On DIV8, cells were washed two times with HBSS before fixation with 4% PFA in PBS. The fixation was performed at RT for 45 min. Then, the cells were incubated with 0.1% Triton X-100 (Sigma-Aldrich) solution for 15 min at RT. Afterwards, cells were washed three times with PBS, followed by an incubation in blocking solution (5% normal horse serum in PBS) for 1 h at RT. Then, primary antibodies, diluted in blocking solution, were added to the cells followed by an incubation overnight at 4\u0026deg;C. The primary antibodies were removed on the next day and the cells were washed three times with PBS. Then, the cells were incubated with the secondary antibodies (diluted in blocking solution) for 2 h at RT in dark to prevent photobleaching. The cells were then washed once with PBS, and were subsequently incubated with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, diluted with PBS at a concentration of 1:10,000) in dark for 20 min at RT. Before mounting, cells were washed three times with PBS to remove all the antibodies and DAPI. Mowiol\u0026reg; (Carl Roth) was used as mounting medium. Images were acquired with the use of a Leica DMi8 fluorescence microscope (Leica, Wetzlar, Germany) with 40x magnification. Images were processed using the Leica Application Suite X software. To quantify the density of the neuronal network, we counted the number of quadruple points per cell by using a modified Neurite Analyzer plugin on the Fiji software [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Each quadruple point is defined as a neuronal junction that contained, or was connected to four different neurites.\u003c/p\u003e\u003cp\u003eThe following primary antibodies were used: rabbit anti-aSyn (1:1000; 701085, Thermo Fisher Scientific); mouse anti beta (III)-tubulin (1:1000; 801202, BioLegend, San Diego, CA, USA). The following secondary antibodies were used: Alexa Fluor 555 Donkey anti-rabbit IgG (1:500; A31572, Thermo Fisher Scientific); Alexa Fluor 647 Donkey anti-mouse IgG (1:500; A32794, Thermo Fisher Scientific).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of activated caspase-3/7 in apoptotic cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCellEvent\u0026trade; Caspase-3/7 Detection Reagent (Thermo Fisher) was used to detect the activation of caspase 3/7 in apoptotic cells. Briefly, the cell culture was performed as mentioned previously. On DIV8, prior the assay, the cell culture medium was removed from the cells. CellEvent\u0026trade; reagent was diluted with pre-warmed HBSS to a final working concentration of 2 \u0026micro;M. The diluted CellEvent\u0026trade; reagent was added to the cells in a dropwise manner to avoid the cell layer from detaching. The cells were incubated with the reagent for 30 min in the cell culture incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e).. After 30 min, the diluted CellEvent\u0026trade; solution was replaced by diluted Hoechst /HBSS solution (final concentration: 1:10000) and was incubated in the cell culture incubator for 5 min. Imaging was performed with a DMi8 live cell imaging fluorescence microcope. The images were processed using the Leica Application Suite X software. The activated caspases signal in the area of the nuclei was quantified by using the Fiji software.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were tested for normality and were analyzed by GraphPad Prism (version 10, GraphPad Software, La Jolla, CA, USA). In all experiments, a minimum of three individual biological replicates were performed. Data were analyzed by multiple t-test if only two experimental groups were present, or by analysis of variance (ANOVA) followed by a post-hoc Bonferroni\u0026rsquo;s test, if at least three experimental groups were present. Statistical significance was assumed if \u003cem\u003ep\u003c/em\u003e values were lower than 0.05. All graphs are presented with error bars indicating the standard error of the mean (SEM). Investigators were not blinded to group allocation during the experiments or outcome assessments. No statistical method was employed to predetermine sample size; instead, sample sizes were determined empirically based on prior experience and observed experimental variability.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e6-OHDA: 6-hydroxydopamine\u003c/p\u003e\n\u003cp\u003eACVR1C: Activin A receptor type 1C\u003c/p\u003e\n\u003cp\u003eALK4: Activin receptor-like kinase 4\u003c/p\u003e\n\u003cp\u003eALK5: Activin receptor-like kinase 5\u003c/p\u003e\n\u003cp\u003eALK7: Activin receptor-like kinase 7\u003c/p\u003e\n\u003cp\u003eANOVA: analysis of variance\u003c/p\u003e\n\u003cp\u003eaSyn: alpha-synuclein\u003c/p\u003e\n\u003cp\u003eBCA: bicinchoninic acid\u003c/p\u003e\n\u003cp\u003eDAPI: 4\u0026apos;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eDIV: days \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDYNLRB1: Dynein light chain roadblock type-1\u003c/p\u003e\n\u003cp\u003eesiRNA: endoribonuclease-prepared siRNA\u003c/p\u003e\n\u003cp\u003eGAPDH: Glyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003eGDNF: Glial cell-derived neurotrophic factor\u003c/p\u003e\n\u003cp\u003eGFP: Green fluorescent protein\u003c/p\u003e\n\u003cp\u003eGSK3B: Glycogen synthase kinase 3 beta\u003c/p\u003e\n\u003cp\u003eGSK8: GSK3B inhibitor-VIII\u003c/p\u003e\n\u003cp\u003eHBSS: Hank\u0026rsquo;s Balanced Salt Solution\u003c/p\u003e\n\u003cp\u003ehuUBQLN1: human ubiquilin 1\u003c/p\u003e\n\u003cp\u003eLBs: Lewy bodies\u003c/p\u003e\n\u003cp\u003eLDH: lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eLUHMES cells: Lund Mesencephalic cells\u003c/p\u003e\n\u003cp\u003eMES: 2-(N-morpholino)ethanesulfonic acid\u003c/p\u003e\n\u003cp\u003eMPP\u003csup\u003e+\u003c/sup\u003e: 1-methyl-4-phenylpyridinium\u003c/p\u003e\n\u003cp\u003eMPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine\u003c/p\u003e\n\u003cp\u003eNADH: nicotinamide adenine dinucleotide\u003c/p\u003e\n\u003cp\u003ePBS: phosphate buffered saline\u003c/p\u003e\n\u003cp\u003ePD: Parkinson\u0026rsquo;s disease\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePFA: paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePOLR2A: RNA Polymerase II Subunit A\u003c/p\u003e\n\u003cp\u003ePVDF: polyvinylidene fluoride\u003c/p\u003e\n\u003cp\u003eSDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003eSMAD2: Mothers against decapentaplegic homolog 2\u003c/p\u003e\n\u003cp\u003eSMAD3: Mothers against decapentaplegic homolog 3\u003c/p\u003e\n\u003cp\u003eSMAD4: Mothers against decapentaplegic homolog 4\u003c/p\u003e\n\u003cp\u003eSN: substantia nigra\u003c/p\u003e\n\u003cp\u003eTBST: Tris-Buffered Saline with 0.1% Tween 20\u003c/p\u003e\n\u003cp\u003eTGFb: Transforming Growth Factor-beta\u003c/p\u003e\n\u003cp\u003eTGFBR1: TGFb type-I receptor\u003c/p\u003e\n\u003cp\u003eTGFBR2: TGFb type-II receptor\u003c/p\u003e\n\u003cp\u003eTH: Tyrosine hydroxylase\u003c/p\u003e\n\u003cp\u003evCSF: ventricular cerebrospinal fluid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: N/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: N/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll datasets used and analyzed in this study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany\u0026rsquo;s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy \u0026ndash; ID 390857198) and within the Hannover Cluster RESIST (EXC 2155 \u0026ndash; - project number 39087428), the German Federal Ministry of Education and Research (BMBF, 01KU1403A EpiPD); the ParkinsonFonds Germany (Hypothesis-free compound screen, alpha-Synuclein fragments in PD); Deutsche Forschungsgemeinschaft (DFG, HO2402/18-1 MSAomics); VolkswagenStiftung (Nieders\u0026auml;chsisches Vorab); Petermax-M\u0026uuml;ller Foundation (Etiology and Therapy of Synucleinopathies and Tauopathies).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eOWC and MH designed the experiments. OWC, LD, SB, VE, and CM performed the experiments. OWC and MH analyzed the data. OWC created the figures and tables and wrote the first draft of the manuscript. All authors critically revised the manuscript. MB, GUH, and MH conceived the study. GUH and MH provided overall project leadership. All authors discussed and commented on the manuscript and agreed to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank Sabine Lang (MHH, Dept. for Neurology) for technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAragon, E., Wang, Q., Zou, Y., Morgani, S. M., Ruiz, L., Kaczmarska, Z., Su, J., Torner, C., Tian, L., Hu, J., Shu, W., Agrawal, S., Gomes, T., Marquez, J. A., Hadjantonakis, A. K., Macias, M. J., \u0026amp; Massague, J. (2019). Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-beta signaling. \u003cem\u003eGenes Dev\u003c/em\u003e,\u003cem\u003e 33\u003c/em\u003e(21-22), 1506-1524. https://doi.org/10.1101/gad.330837.119 \u003c/li\u003e\n\u003cli\u003eChakroun, T., Evsyukov, V., Nykanen, N. P., Hollerhage, M., Schmidt, A., Kamp, F., Ruf, V. C., Wurst, W., Rosler, T. W., \u0026amp; Hoglinger, G. U. (2020). Alpha-synuclein fragments trigger distinct aggregation pathways. \u003cem\u003eCell Death Dis\u003c/em\u003e,\u003cem\u003e 11\u003c/em\u003e(2), 84. https://doi.org/10.1038/s41419-020-2285-7 \u003c/li\u003e\n\u003cli\u003eChia, S. J., Tan, E. K., \u0026amp; Chao, Y. X. (2020). Historical Perspective: Models of Parkinson\u0026apos;s Disease. \u003cem\u003eInt J Mol Sci\u003c/em\u003e,\u003cem\u003e 21\u003c/em\u003e(7). https://doi.org/10.3390/ijms21072464 \u003c/li\u003e\n\u003cli\u003eDecressac, M., Mattsson, B., \u0026amp; Bjorklund, A. (2012). Comparison of the behavioural and histological characteristics of the 6-OHDA and alpha-synuclein rat models of Parkinson\u0026apos;s disease. \u003cem\u003eExp Neurol\u003c/em\u003e,\u003cem\u003e 235\u003c/em\u003e(1), 306-315. https://doi.org/10.1016/j.expneurol.2012.02.012 \u003c/li\u003e\n\u003cli\u003eDecressac, M., Ulusoy, A., Mattsson, B., Georgievska, B., Romero-Ramos, M., Kirik, D., \u0026amp; Bjorklund, A. (2011). GDNF fails to exert neuroprotection in a rat alpha-synuclein model of Parkinson\u0026apos;s disease. \u003cem\u003eBrain\u003c/em\u003e,\u003cem\u003e 134\u003c/em\u003e(Pt 8), 2302-2311. https://doi.org/10.1093/brain/awr149 \u003c/li\u003e\n\u003cli\u003eDobolyi, A., Vincze, C., Pal, G., \u0026amp; Lovas, G. (2012). The neuroprotective functions of transforming growth factor beta proteins. \u003cem\u003eInt J Mol Sci\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e(7), 8219-8258. https://doi.org/10.3390/ijms13078219 \u003c/li\u003e\n\u003cli\u003eDong, Y., Lu, J., Zhang, S., Chen, L., Wen, J., Wang, F., Mao, Y., Li, L., Zhang, J., Liao, S., \u0026amp; Dong, L. (2023). Design, synthesis and bioevaluation of 1,2,4-thiadiazolidine-3,5-dione derivatives as potential GSK-3beta inhibitors for the treatment of Alzheimer\u0026apos;s disease. \u003cem\u003eBioorg Chem\u003c/em\u003e,\u003cem\u003e 134\u003c/em\u003e, 106446. https://doi.org/10.1016/j.bioorg.2023.106446 \u003c/li\u003e\n\u003cli\u003eEmin, D., Zhang, Y. P., Lobanova, E., Miller, A., Li, X., Xia, Z., Dakin, H., Sideris, D. I., Lam, J. Y. L., Ranasinghe, R. T., Kouli, A., Zhao, Y., De, S., Knowles, T. P. J., Vendruscolo, M., Ruggeri, F. S., Aigbirhio, F. I., Williams-Gray, C. H., \u0026amp; Klenerman, D. (2022). Small soluble alpha-synuclein aggregates are the toxic species in Parkinson\u0026apos;s disease. \u003cem\u003eNat Commun\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e(1), 5512. https://doi.org/10.1038/s41467-022-33252-6 \u003c/li\u003e\n\u003cli\u003eEngebretsen, K. V., Skardal, K., Bjornstad, S., Marstein, H. S., Skrbic, B., Sjaastad, I., Christensen, G., Bjornstad, J. L., \u0026amp; Tonnessen, T. (2014). Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5. \u003cem\u003eJ Mol Cell Cardiol\u003c/em\u003e,\u003cem\u003e 76\u003c/em\u003e, 148-157. https://doi.org/10.1016/j.yjmcc.2014.08.008 \u003c/li\u003e\n\u003cli\u003eGash, D. M., Zhang, Z., Ovadia, A., Cass, W. A., Yi, A., Simmerman, L., Russell, D., Martin, D., Lapchak, P. A., Collins, F., Hoffer, B. J., \u0026amp; Gerhardt, G. A. (1996). Functional recovery in parkinsonian monkeys treated with GDNF. \u003cem\u003eNature\u003c/em\u003e,\u003cem\u003e 380\u003c/em\u003e(6571), 252-255. https://doi.org/10.1038/380252a0 \u003c/li\u003e\n\u003cli\u003eGuo, X., Ramirez, A., Waddell, D. S., Li, Z., Liu, X., \u0026amp; Wang, X. F. (2008). Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling. \u003cem\u003eGenes Dev\u003c/em\u003e,\u003cem\u003e 22\u003c/em\u003e(1), 106-120. https://doi.org/10.1101/gad.1590908 \u003c/li\u003e\n\u003cli\u003eHaas, A. J., Prigent, S., Dutertre, S., Le Drean, Y., \u0026amp; Le Page, Y. (2016). Neurite analyzer: An original Fiji plugin for quantification of neuritogenesis in two-dimensional images. \u003cem\u003eJ Neurosci Methods\u003c/em\u003e,\u003cem\u003e 271\u003c/em\u003e, 86-91. https://doi.org/10.1016/j.jneumeth.2016.07.011 \u003c/li\u003e\n\u003cli\u003eHata, A., \u0026amp; Chen, Y. G. (2016). TGF-beta Signaling from Receptors to Smads. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(9). https://doi.org/10.1101/cshperspect.a022061 \u003c/li\u003e\n\u003cli\u003eHeldin, C. H., \u0026amp; Moustakas, A. (2016). Signaling Receptors for TGF-beta Family Members. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(8). https://doi.org/10.1101/cshperspect.a022053 \u003c/li\u003e\n\u003cli\u003eHill, C. S. (2016). Transcriptional Control by the SMADs. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e,\u003cem\u003e 8\u003c/em\u003e(10). https://doi.org/10.1101/cshperspect.a022079 \u003c/li\u003e\n\u003cli\u003eHoglinger, G. U., Adler, C. H., Berg, D., Klein, C., Outeiro, T. F., Poewe, W., Postuma, R., Stoessl, A. J., \u0026amp; Lang, A. E. (2024). A biological classification of Parkinson\u0026apos;s disease: the SynNeurGe research diagnostic criteria. \u003cem\u003eLancet Neurol\u003c/em\u003e,\u003cem\u003e 23\u003c/em\u003e(2), 191-204. https://doi.org/10.1016/S1474-4422(23)00404-0 \u003c/li\u003e\n\u003cli\u003eHoglinger, G. U., Huppertz, H. J., Wagenpfeil, S., Andres, M. V., Belloch, V., Leon, T., Del Ser, T., \u0026amp; Investigators, T. M. (2014). Tideglusib reduces progression of brain atrophy in progressive supranuclear palsy in a randomized trial. \u003cem\u003eMov Disord\u003c/em\u003e,\u003cem\u003e 29\u003c/em\u003e(4), 479-487. https://doi.org/10.1002/mds.25815 \u003c/li\u003e\n\u003cli\u003eH\u0026ouml;llerhage, M., Duan, L., Chua, OWH., Moebius, C., Bothe, S., Hopfner, F., Wahl-Schott., C., Bickle., M., H\u0026ouml;glinger., GU. (2025). A genome-wide RNA interference screening reveals protectiveness of SNX5 knockdown in a Parkinson\u0026rsquo;s disease cell model. Transl Neurodegener. 2025 Jun 3;14(1):27. https://doi.org/10.1186/s40035-025-00486-5.\u003c/li\u003e\n\u003cli\u003eHollerhage, M., Goebel, J. N., de Andrade, A., Hildebrandt, T., Dolga, A., Culmsee, C., Oertel, W. H., Hengerer, B., \u0026amp; Hoglinger, G. U. (2014). Trifluoperazine rescues human dopaminergic cells from wild-type alpha-synuclein-induced toxicity. \u003cem\u003eNeurobiol Aging\u003c/em\u003e,\u003cem\u003e 35\u003c/em\u003e(7), 1700-1711. https://doi.org/10.1016/j.neurobiolaging.2014.01.027 \u003c/li\u003e\n\u003cli\u003eHollerhage, M., Stepath, M., Kohl, M., Pfeiffer, K., Chua, O. W. H., Duan, L., Hopfner, F., Eisenacher, M., Marcus, K., \u0026amp; Hoglinger, G. U. (2022). Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein. \u003cem\u003eFront Neurol\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e, 787059. https://doi.org/10.3389/fneur.2022.787059 \u003c/li\u003e\n\u003cli\u003eJin, Q., Ding, W., \u0026amp; Mulder, K. M. (2007). Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway. \u003cem\u003eJ Biol Chem\u003c/em\u003e,\u003cem\u003e 282\u003c/em\u003e(26), 19122-19132. https://doi.org/10.1074/jbc.M609915200 \u003c/li\u003e\n\u003cli\u003eJornvall, H., Blokzijl, A., ten Dijke, P., \u0026amp; Ibanez, C. F. (2001). The orphan receptor serine/threonine kinase ALK7 signals arrest of proliferation and morphological differentiation in a neuronal cell line. \u003cem\u003eJ Biol Chem\u003c/em\u003e,\u003cem\u003e 276\u003c/em\u003e(7), 5140-5146. https://doi.org/10.1074/jbc.M005200200 \u003c/li\u003e\n\u003cli\u003eJornvall, H., Reissmann, E., Andersson, O., Mehrkash, M., \u0026amp; Ibanez, C. F. (2004). ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse. \u003cem\u003eMol Cell Biol\u003c/em\u003e,\u003cem\u003e 24\u003c/em\u003e(21), 9383-9389. https://doi.org/10.1128/MCB.24.21.9383-9389.2004 \u003c/li\u003e\n\u003cli\u003eKang, H., Seo, E., Oh, Y. S., \u0026amp; Jun, H. S. (2022). TGF-beta activates NLRP3 inflammasome by an autocrine production of TGF-beta in LX-2 human hepatic stellate cells. \u003cem\u003eMol Cell Biochem\u003c/em\u003e,\u003cem\u003e 477\u003c/em\u003e(5), 1329-1338. https://doi.org/10.1007/s11010-022-04369-5 \u003c/li\u003e\n\u003cli\u003eKomai, T., Inoue, M., Okamura, T., Morita, K., Iwasaki, Y., Sumitomo, S., Shoda, H., Yamamoto, K., \u0026amp; Fujio, K. (2018). Transforming Growth Factor-beta and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals. \u003cem\u003eFront Immunol\u003c/em\u003e,\u003cem\u003e 9\u003c/em\u003e, 1364. https://doi.org/10.3389/fimmu.2018.01364 \u003c/li\u003e\n\u003cli\u003eKrieglstein, K. (2004). Factors promoting survival of mesencephalic dopaminergic neurons. \u003cem\u003eCell Tissue Res\u003c/em\u003e,\u003cem\u003e 318\u003c/em\u003e(1), 73-80. https://doi.org/10.1007/s00441-004-0920-8 \u003c/li\u003e\n\u003cli\u003eKrieglstein, K., Miyazono, K., ten Dijke, P., \u0026amp; Unsicker, K. (2012). TGF-beta in aging and disease. \u003cem\u003eCell Tissue Res\u003c/em\u003e,\u003cem\u003e 347\u003c/em\u003e(1), 5-9. https://doi.org/10.1007/s00441-011-1278-3 \u003c/li\u003e\n\u003cli\u003eKrieglstein, K., Suter-Crazzolara, C., Fischer, W. H., \u0026amp; Unsicker, K. (1995). TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. \u003cem\u003eEMBO J\u003c/em\u003e,\u003cem\u003e 14\u003c/em\u003e(4), 736-742. https://doi.org/10.1002/j.1460-2075.1995.tb07052.x \u003c/li\u003e\n\u003cli\u003eKrieglstein, K., Suter-Crazzolara, C., \u0026amp; Unsicker, K. (1995). Development of mesencephalic dopaminergic neurons and the transforming growth factor-beta superfamily. \u003cem\u003eJ Neural Transm Suppl\u003c/em\u003e,\u003cem\u003e 46\u003c/em\u003e, 209-216. https://www.ncbi.nlm.nih.gov/pubmed/8821057 \u003c/li\u003e\n\u003cli\u003eKrieglstein, K., Zheng, F., Unsicker, K., \u0026amp; Alzheimer, C. (2011). More than being protective: functional roles for TGF-beta/activin signaling pathways at central synapses. \u003cem\u003eTrends Neurosci\u003c/em\u003e,\u003cem\u003e 34\u003c/em\u003e(8), 421-429. https://doi.org/10.1016/j.tins.2011.06.002 \u003c/li\u003e\n\u003cli\u003eLi, J., Ma, S., Chen, J., Hu, K., Li, Y., Zhang, Z., Su, Z., Woodgett, J. R., Li, M., \u0026amp; Huang, Q. (2020). GSK-3beta Contributes to Parkinsonian Dopaminergic Neuron Death: Evidence From Conditional Knockout Mice and Tideglusib. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e,\u003cem\u003e 13\u003c/em\u003e, 81. https://doi.org/10.3389/fnmol.2020.00081 \u003c/li\u003e\n\u003cli\u003eLiu, L., Liu, X., Ren, X., Tian, Y., Chen, Z., Xu, X., Du, Y., Jiang, C., Fang, Y., Liu, Z., Fan, B., Zhang, Q., Jin, G., Yang, X., \u0026amp; Zhang, X. (2016). Smad2 and Smad3 have differential sensitivity in relaying TGFbeta signaling and inversely regulate early lineage specification. \u003cem\u003eSci Rep\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e, 21602. https://doi.org/10.1038/srep21602 \u003c/li\u003e\n\u003cli\u003eLiu, Q., Qiu, J., Liang, M., Golinski, J., van Leyen, K., Jung, J. E., You, Z., Lo, E. H., Degterev, A., \u0026amp; Whalen, M. J. (2014). Akt and mTOR mediate programmed necrosis in neurons. \u003cem\u003eCell Death Dis\u003c/em\u003e,\u003cem\u003e 5\u003c/em\u003e(2), e1084. https://doi.org/10.1038/cddis.2014.69 \u003c/li\u003e\n\u003cli\u003eLoomans, H. A., \u0026amp; Andl, C. D. (2016). Activin receptor-like kinases: a diverse family playing an important role in cancer. \u003cem\u003eAm J Cancer Res\u003c/em\u003e,\u003cem\u003e 6\u003c/em\u003e(11), 2431-2447. https://www.ncbi.nlm.nih.gov/pubmed/27904762 \u003c/li\u003e\n\u003cli\u003eLotharius, J., Barg, S., Wiekop, P., Lundberg, C., Raymon, H. K., \u0026amp; Brundin, P. (2002). Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. \u003cem\u003eJ Biol Chem\u003c/em\u003e,\u003cem\u003e 277\u003c/em\u003e(41), 38884-38894. https://doi.org/10.1074/jbc.M205518200 \u003c/li\u003e\n\u003cli\u003eLotharius, J., Falsig, J., van Beek, J., Payne, S., Dringen, R., Brundin, P., \u0026amp; Leist, M. (2005). Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. \u003cem\u003eJ Neurosci\u003c/em\u003e,\u003cem\u003e 25\u003c/em\u003e(27), 6329-6342. https://doi.org/10.1523/JNEUROSCI.1746-05.2005 \u003c/li\u003e\n\u003cli\u003eLovestone, S., Boada, M., Dubois, B., Hull, M., Rinne, J. O., Huppertz, H. J., Calero, M., Andres, M. V., Gomez-Carrillo, B., Leon, T., del Ser, T., \u0026amp; investigators, A. (2015). A phase II trial of tideglusib in Alzheimer\u0026apos;s disease. \u003cem\u003eJ Alzheimers Dis\u003c/em\u003e,\u003cem\u003e 45\u003c/em\u003e(1), 75-88. https://doi.org/10.3233/JAD-141959 \u003c/li\u003e\n\u003cli\u003eMogi, M., Harada, M., Kondo, T., Narabayashi, H., Riederer, P., \u0026amp; Nagatsu, T. (1995). Transforming growth factor-beta 1 levels are elevated in the striatum and in ventricular cerebrospinal fluid in Parkinson\u0026apos;s disease. \u003cem\u003eNeurosci Lett\u003c/em\u003e,\u003cem\u003e 193\u003c/em\u003e(2), 129-132. https://doi.org/10.1016/0304-3940(95)11686-q \u003c/li\u003e\n\u003cli\u003eRausch, M. P., Hahn, T., Ramanathapuram, L., Bradley-Dunlop, D., Mahadevan, D., Mercado-Pimentel, M. E., Runyan, R. B., Besselsen, D. G., Zhang, X., Cheung, H. K., Lee, W. C., Ling, L. E., \u0026amp; Akporiaye, E. T. (2009). An orally active small molecule TGF-beta receptor I antagonist inhibits the growth of metastatic murine breast cancer. \u003cem\u003eAnticancer Res\u003c/em\u003e,\u003cem\u003e 29\u003c/em\u003e(6), 2099-2109. https://www.ncbi.nlm.nih.gov/pubmed/19528470 \u003c/li\u003e\n\u003cli\u003eRichter, F., Stanojlovic, M., Kaufer, C., Gericke, B., \u0026amp; Feja, M. (2023). A Mouse Model to Test Novel Therapeutics for Parkinson\u0026apos;s Disease: an Update on the Thy1-aSyn (\u0026quot;line 61\u0026quot;) Mice. \u003cem\u003eNeurotherapeutics\u003c/em\u003e. https://doi.org/10.1007/s13311-022-01338-0 \u003c/li\u003e\n\u003cli\u003eRunyan, C. E., Schnaper, H. W., \u0026amp; Poncelet, A. C. (2005). The role of internalization in transforming growth factor beta1-induced Smad2 association with Smad anchor for receptor activation (SARA) and Smad2-dependent signaling in human mesangial cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e,\u003cem\u003e 280\u003c/em\u003e(9), 8300-8308. https://doi.org/10.1074/jbc.M407939200 \u003c/li\u003e\n\u003cli\u003eSanchez-Capelo, A., Colin, P., Guibert, B., Biguet, N. F., \u0026amp; Mallet, J. (2003). Transforming growth factor beta1 overexpression in the nigrostriatal system increases the dopaminergic deficit of MPTP mice. \u003cem\u003eMol Cell Neurosci\u003c/em\u003e,\u003cem\u003e 23\u003c/em\u003e(4), 614-625. https://doi.org/10.1016/s1044-7431(03)00081-2 \u003c/li\u003e\n\u003cli\u003eSpillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., \u0026amp; Goedert, M. (1998). alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson\u0026apos;s disease and dementia with lewy bodies. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e,\u003cem\u003e 95\u003c/em\u003e(11), 6469-6473. https://doi.org/10.1073/pnas.95.11.6469 \u003c/li\u003e\n\u003cli\u003eSpillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., \u0026amp; Goedert, M. (1997). Alpha-synuclein in Lewy bodies. \u003cem\u003eNature\u003c/em\u003e,\u003cem\u003e 388\u003c/em\u003e(6645), 839-840. https://doi.org/10.1038/42166 \u003c/li\u003e\n\u003cli\u003eSuzuki, E., Kim, S., Cheung, H. K., Corbley, M. J., Zhang, X., Sun, L., Shan, F., Singh, J., Lee, W. C., Albelda, S. M., \u0026amp; Ling, L. E. (2007). A novel small-molecule inhibitor of transforming growth factor beta type I receptor kinase (SM16) inhibits murine mesothelioma tumor growth in vivo and prevents tumor recurrence after surgical resection. \u003cem\u003eCancer Res\u003c/em\u003e,\u003cem\u003e 67\u003c/em\u003e(5), 2351-2359. https://doi.org/10.1158/0008-5472.CAN-06-2389 \u003c/li\u003e\n\u003cli\u003eTesseur, I., Nguyen, A., Chang, B., Li, L., Woodling, N. S., Wyss-Coray, T., \u0026amp; Luo, J. (2017). Deficiency in Neuronal TGF-beta Signaling Leads to Nigrostriatal Degeneration and Activation of TGF-beta Signaling Protects against MPTP Neurotoxicity in Mice. \u003cem\u003eJ Neurosci\u003c/em\u003e,\u003cem\u003e 37\u003c/em\u003e(17), 4584-4592. https://doi.org/10.1523/JNEUROSCI.2952-16.2017 \u003c/li\u003e\n\u003cli\u003eTolosa, E., Garrido, A., Scholz, S. W., \u0026amp; Poewe, W. (2021). Challenges in the diagnosis of Parkinson\u0026apos;s disease. \u003cem\u003eLancet Neurol\u003c/em\u003e,\u003cem\u003e 20\u003c/em\u003e(5), 385-397. https://doi.org/10.1016/S1474-4422(21)00030-2 \u003c/li\u003e\n\u003cli\u003eTomac, A., Lindqvist, E., Lin, L. F., Ogren, S. O., Young, D., Hoffer, B. J., \u0026amp; Olson, L. (1995). Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. \u003cem\u003eNature\u003c/em\u003e,\u003cem\u003e 373\u003c/em\u003e(6512), 335-339. https://doi.org/10.1038/373335a0 \u003c/li\u003e\n\u003cli\u003eTsuchida, K., Sawchenko, P. E., Nishikawa, S., \u0026amp; Vale, W. W. (1996). Molecular cloning of a novel type I receptor serine/threonine kinase for the TGF beta superfamily from rat brain. \u003cem\u003eMol Cell Neurosci\u003c/em\u003e,\u003cem\u003e 7\u003c/em\u003e(6), 467-478. https://doi.org/10.1006/mcne.1996.0034 \u003c/li\u003e\n\u003cli\u003eTsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L., \u0026amp; Wrana, J. L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. \u003cem\u003eCell\u003c/em\u003e,\u003cem\u003e 95\u003c/em\u003e(6), 779-791. https://doi.org/10.1016/s0092-8674(00)81701-8 \u003c/li\u003e\n\u003cli\u003eVawter, M. P., Dillon-Carter, O., Tourtellotte, W. W., Carvey, P., \u0026amp; Freed, W. J. (1996). TGFbeta1 and TGFbeta2 concentrations are elevated in Parkinson\u0026apos;s disease in ventricular cerebrospinal fluid. \u003cem\u003eExp Neurol\u003c/em\u003e,\u003cem\u003e 142\u003c/em\u003e(2), 313-322. https://doi.org/10.1006/exnr.1996.0200 \u003c/li\u003e\n\u003cli\u003eWhone, A., Luz, M., Boca, M., Woolley, M., Mooney, L., Dharia, S., Broadfoot, J., Cronin, D., Schroers, C., Barua, N. U., Longpre, L., Barclay, C. L., Boiko, C., Johnson, G. A., Fibiger, H. C., Harrison, R., Lewis, O., Pritchard, G., Howell, M., . . . Gill, S. S. (2019). Randomized trial of intermittent intraputamenal glial cell line-derived neurotrophic factor in Parkinson\u0026apos;s disease. \u003cem\u003eBrain\u003c/em\u003e,\u003cem\u003e 142\u003c/em\u003e(3), 512-525. https://doi.org/10.1093/brain/awz023 \u003c/li\u003e\n\u003cli\u003eWong, Y. C., \u0026amp; Krainc, D. (2017). alpha-synuclein toxicity in neurodegeneration: mechanism and therapeutic strategies. \u003cem\u003eNat Med\u003c/em\u003e,\u003cem\u003e 23\u003c/em\u003e(2), 1-13. https://doi.org/10.1038/nm.4269 \u003c/li\u003e\n\u003cli\u003eZhang, Y., Alexander, P. B., \u0026amp; Wang, X. F. (2017). TGF-beta Family Signaling in the Control of Cell Proliferation and Survival. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e,\u003cem\u003e 9\u003c/em\u003e(4). https://doi.org/10.1101/cshperspect.a022145 \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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, alpha-synuclein, genome-wide RNAi screening, Transforming Growth Factor-beta signaling","lastPublishedDoi":"10.21203/rs.3.rs-6867964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6867964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is histopathologically defined by the presence of Lewy bodies, which are intracellular proteinaceous inclusions that contain mainly aggregated alpha-synuclein (aSyn). It is believed that oligomeric intermediates between monomeric aSyn and large aggregates are neurotoxic, which would lead to the demise of dopaminergic neurons. Therefore, novel therapies preventing aSyn-induced cell death need to be developed.\u003c/p\u003e\u003cp\u003eTherefore, we performed a genome-wide siRNA screening in an aSyn-induced dopaminergic cell death model and found the knockdown of three Transforming Growth Factor-beta (TGFb) pathway-related genes to be protective. Hence, we hypothesized that a reduction in TGFb signaling would protect dopaminergic neurons from aSyn-induced toxicity. Thus, we validated the results of the genome-wide knockdown screening with the use of two different types of siRNAs. We confirmed that the knockdown of Activin receptor-like kinase 5 (\u003cem\u003eALK5\u003c/em\u003e) and Mothers against decapentaplegic homolog 2 (\u003cem\u003eSMAD2\u003c/em\u003e), two genes of the TGFb pathway, protected dopaminergic neurons from aSyn-induced toxicity. An increase in TGFb signaling by treatment with TGFb ligands further exacerbated aSyn-induced toxicity, whereas this effect was mitigated by knockdown of \u003cem\u003eALK5\u003c/em\u003e, \u003cem\u003eSMAD2\u003c/em\u003e, or Dynein light chain roadblock type-1 (\u003cem\u003eDYNLRB1\u003c/em\u003e). Moreover, TGFb ligand treatment induced an up-regulation of \u003cem\u003eSNCA\u003c/em\u003e mRNA expression in aSyn-overexpressing cells. Interestingly, consistent with the literature, we identified an up-regulation of the genes of the TGFb pathway in aSyn-overexpressing cells.\u003c/p\u003e\u003cp\u003eAltogether, we identified a potential protective role of the TGFb pathway against aSyn-induced toxicity. These findings provide a rationale for the development of novel strategies against PD.\u003c/p\u003e","manuscriptTitle":"Inhibition of TGF-beta signaling protects from alpha-synuclein induced toxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-27 11:15:05","doi":"10.21203/rs.3.rs-6867964/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16cfa099-e1f7-4007-8812-7844571da6f7","owner":[],"postedDate":"July 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50523009,"name":"Health sciences/Diseases/Neurological disorders/Parkinson's disease"},{"id":50523010,"name":"Biological sciences/Molecular biology/RNAi"},{"id":50523011,"name":"Biological sciences/Cell biology/Cell signalling/Growth factor signalling"}],"tags":[],"updatedAt":"2026-01-24T08:10:30+00:00","versionOfRecord":{"articleIdentity":"rs-6867964","link":"https://doi.org/10.1038/s41420-025-02901-2","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2025-12-12 05:00:00","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-07-27 11:15:05","video":"","vorDoi":"10.1038/s41420-025-02901-2","vorDoiUrl":"https://doi.org/10.1038/s41420-025-02901-2","workflowStages":[]},"version":"v1","identity":"rs-6867964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6867964","identity":"rs-6867964","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}