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[Preprint]. 2024 Oct 17:2024.04.18.590103. Originally published 2024 Apr 21. [Version 2] doi: 10.1101/2024.04.18.590103

SON-dependent nuclear speckle rejuvenation alleviates proteinopathies

William Dion 1,#, Yuren Tao 2,#, Maci Chambers 1, Shanshan Zhao 2, Riley K Arbuckle 3,4, Michelle Sun 1, Syeda Kubra 1, Imran Jamal 1, Yuhang Nie 2, Megan Ye 1, Mads B Larsen 1, Daniel Camarco 1, Eleanor Ickes 1, Claire DuPont 1, Haokun Wang 1, Bingjie Wang 3, Silvia Liu 5,6, Shaohua Pi 3, Bill B Chen 1,7, Yuanyuan Chen 3,8,*, Xu Chen 2,*, Bokai Zhu 1,5,9,*
PMCID: PMC11042303  PMID: 38659924

Abstract

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as a pivotal membrane-less organelle responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate alleviated retina degeneration and reduced tauopathy by promoting autophagy and ubiquitin-proteasome system in a SON-dependent manner without causing cellular stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

Introduction:

Proteinopathies are diseases associated with the accumulation of misfolded proteins, which often arise from a decline in proteostasis pathways, including the ubiquitin-proteasome system (UPS), the ER-Golgi protein secretory pathways, and autophagy lysosomal pathway (ALP)1, 2. However, therapies targeting singular pathways have limited efficacy, indicating an incomplete understanding of disease mechanisms.

We recently discovered that under physiological conditions, the network of proteostasis pathways manifests as cell-autonomous 12-hour (12h) ultradian rhythms, regulated by a dedicated 12h oscillator, independent from the 24h circadian clock and the cell cycle3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. By studying this 12h oscillator, we uncovered an unexpected role of nuclear speckles in global proteostasis control9. Nuclear speckles are membrane-less organelles important for mRNA processing and gene regulation14, 15, 16, 17, 18, and their liquid-liquid phase separation (LLPS) dynamics dictate the global transcriptional capacity of proteostasis genes9. Moderate overexpression of the nuclear speckle scaffolding protein SON is sufficient to decrease nuclear speckle sphericity, increase the recruitment of nuclear speckles to chromatin, amplify proteostasis gene expression, and reduce protein aggregation9. Conversely, reducing SON level leads to much more spherical and stagnant speckles, sequesters nuclear speckles away from chromatin, blunts proteostasis gene expression and subsequently elevates intracellular protein aggregates19. More importantly, SON expression decreases with age in various tissues in mice, concomitant with more spherical and smaller nuclear speckles (Supplementary Fig. 1af). In addition, reduced SON expression was also observed in aging human lungs, and in brain tissues of human subjects with Alzheimer’s disease (Supplementary Fig. 1gi)9, 20. Based upon these results, we herein propose that enhancing SON expression or function, either genetically or pharmacologically, under aging and disease conditions, could potentially restore nuclear speckle morphology and function. This, in turn, would bolster the entire protein quality control system, thereby delaying or even reversing the progression of both aging-related and inherited proteinopathies. We introduce this concept as SON-dependent ‘nuclear speckle rejuvenation’ (Fig. 1a).

Fig. 1. Genetic rejuvenation of nuclear speckle transcriptionally reprograms global proteostasis and YAP1 signaling in an opposing manner.

Fig. 1.

(a) The diagram of the approach of nuclear speckle rejuvenation to alleviate proteinopathy. (b) PCA of global transcriptional response to Tu in the presence of SON OE (via CRISPRa) or KD (via siRNA). (c, d) Relative expression (R.E.) of representative proteostasis genes (c) and YAP1 target genes (d) in response to Tu in the presence of SON OE/KD (n=4 for SON KD, and n=3 for SON OE). (e) Heat map of relative fold change of gene expression by Tu in SON OE/KD cells compared to control cells. Only those genes with at least 1.4-fold induction by Tu (log 2 > 0.5) with a p value smaller than 0.05 in control condition are included. Among these genes are 461 genes whose induction by Tu are further amplified by SON OE (induced more) and dampened (induced less) by SON KD; and 901 genes whose repression by Tu are further amplified by Son OE (repressed further) and dampened (repressed less) by SON KD. (f) GO analysis of those 461 and 901 genes showing enriched KEGG pathways. (g) Enriched XBP1s binding motif ACGTCA on the promoters of 461 genes. (h) Top enriched GO terms for top 500 most abundant proteins that are detected in hepatic XBP1s interactome at CT8, as previously reported10. (i) Heatmap showing relative abundance of 45 proteins involved in mRNA splicing and processing within the XBP1s interactome at CT0 and CT8, respectively. (j) Enriched TEAD2 binding motif GGCGG on the promoters of 901 genes. Western blot (k) and quantification (l) of nuclear and cytosolic level of YAP1 in control and SON OE cells in response to Tu (n=2). Scratch assay with representative images (m) and quantification (n) of cell migration rate in control and SON OE cells in response to Tu (n=5). All data mean ± standard error of the mean (S.E.M.). Statistical tests used: unpaired one-tailed Student’s t-test for c, d, l, and n. Paired one-tailed Student’s t-test for i.

In this study, we initially characterized the comprehensive transcriptome changes resulting from SON-mediated nuclear speckle rejuvenation and unexpectedly discovered a broader proteostasis framework that incorporates SON-mediated nuclear speckles condensation, unfolded protein response (UPR) transcription factors (TF)-mediated proteostasis gene activation and repression of YAP1 transcriptional activity. Via a high-throughput drug screen, we identified pyrvinium pamoate (PP) as a small molecule nuclear speckle rejuvenator that recapitulates the entire transcriptome changes elicited by SON overexpression. Mechanistically, via a cell-free nuclear speckle reconstitution system, we demonstrated that PP exerts transcriptional reprogramming via reducing the surface/interfacial tension of nuclear speckle condensates and promoting their wetting of genomic DNA via targeting the intrinsically disordered region (IDR) of SON. In preclinical models, PP exhibited strong efficacy in protecting against both tauopathy and retinal degeneration at nanomolar concentration without inducing cellular stress. Lastly, we showed that both the reduction of protein quality control gene expression and increase of YAP1 transcriptional activity is associated with retinal degeneration in mice and tauopathy in humans.

Result:

Genetic rejuvenation of nuclear speckles transcriptionally reprograms global proteostasis and YAP1 activity in an opposing manner.

To determine the extent by which SON transcriptionally reprograms gene expression under both basal and proteotoxic stress conditions, we performed bulk mRNA-Seq on immortalized mouse embryonic fibroblasts (MEFs) with either SON knockdown (KD) by siRNA or overexpression (OE) via CRISPRa21, in the absence or presence of the ER stress inducer tunicamycin (Tu) as previously described (Supplementary Table1)9. Principal component analysis (PCA) on total mRNA level indicated that while SON manipulation has little effects on global gene expression under basal condition, SON OE and KD significantly amplified and dampened the global transcriptional response to ER stress, respectively (Fig. 1b). These include 461 genes that are normally induced, and 901 genes repressed by Tu under normal SON expression condition (Fig. 1ce, Supplementary Fig. 2a). For both groups of genes, we further observed a strong correlation between the relative fold induction or repression for each gene under SON OE and KD conditions (Supplementary Fig. 2b), further demonstrating the robustness of bidirectional control on proteostasis gene expression by SON.

As expected, gene ontology (GO) analysis revealed that those ER stress-induced 461 genes are strongly enriched in protein quality control pathways, including protein folding (such as Pdia3, Dnajb11 and Manf), ER/Golgi quality control (such as Sec23b, Hyou1 and Hspa5), tRNA aminoacylation (such as Gars, Iars and Eprs), ER-associated protein degradation (ERAD) (such as Edem1, Syvn1, Sel1l and Ube2g2), and autophagy (such as Sqstm1 and Atg13) (Fig. 1f, Supplementary Fig. 2a). To shed light on the mechanisms by which SON transcriptionally amplifies gene activation in response to ER stress, we performed both Landscape In Silico deletion Analysis (LISA)22 and motif analysis to infer the transcriptional regulators that may mediate nuclear speckles interactions with chromatin. Both analyses revealed basic leucine zipper (bZIP) transcription factors (TFs), including ATF6, XBP1, ATF4 and CREB1 as the top candidates (Fig. 1g and Supplementary Fig. 2c). ChIP-qPCR further showed increased recruitment of nuclear speckles to the 3’ regions of selective proteostasis genes in response to SON OE under both basal and ER stress conditions, concomitant with increased recruitment of XBP1s to the promoter regions of the same genes (Supplementary Fig. 2d, e). To corroborate our in vitro findings, we further examined a recently published murine in vivo hepatic XBP1s interactome dataset10, and found proteins involved in mRNA splicing and processing are very strongly enriched in the XBP1s interactome at CT8, a time when hepatic SON expression peaks9, with PRPF8, SNRNP200 and DHX9 being the top three most abundant proteins detected in the entire XBP1s interactome at CT8 (Fig. 1h, i). By contrast, at CT0 when SON expression is the lowest, the amount of splicing proteins that interact with XBP1s is markedly reduced (Fig. 1i). The observed decreased recruitment of splicing proteins to XBP1s at CT0 is not due to reduced XBP1s level itself, as the hepatic XBP1s expression at CT0 is in fact higher compared to CT83, 23. These results thus reinforce the notion that nuclear speckles rejuvenation by SON OE is sufficient to amplify the global proteostasis transcriptional activation, likely via facilitating physical interactions between nuclear speckles and UPR TF like XBP1s.

Compared to induced genes, much less is known about the gene programs that are repressed under ER stress. GO analysis indicate that those 901 ER stress-repressed genes are strongly enriched in Hippo-YAP1 signaling that regulates the diverse biological processes of angiogenesis, axon guidance, epithelial to mesenchymal transition (EMT), wound healing, cell adhesion, cell migration, and extra cellular matrix organization, with examples of canonical YAP1 target genes Bmp4, Tuba1a, Fzd2, Tgfb3 and Yap1 itself and its transcriptional partner Tead224, 25, 26 (Fig. 1cf and Supplementary Fig. 2a). YAP1 and TEAD2 were further predicted to be transcriptional regulators of these 901 ER stress-repressed genes via both LISA and motif analysis (Fig. 1j and Supplementary Fig. 2c) and the nuclear YAP1 level was significantly reduced in response to either Tu or SON OE and further decreased upon the combination of the two (Fig. 1k, l). We further performed TEAD luciferase reporter assay and found that both SON OE and Tu significantly reduced the TEAD response element-driven luciferase activity in MEFs, with the lowest observed in SON OE cells under ER stress (Supplementary Fig. 3a). Scratch assays further confirmed that both SON OE and Tu significantly reduced cell migration in MEFs, with the lowest observed in SON OE cells under ER stress (Fig. 1m, n). To rule out the possibility that the global repression of YAP1 transcriptional output during ER stress is specific to MEFs or Tu, we further analyzed a recent transcriptome dataset in the human astrocytoma-derived LN-308 cell line in response to both Tu and thapsigargin (Thap) (another ER stress inducer) treatments27, and observed a strong downregulation of genes involved in YAP1 signaling under both treatments that progressed with time (Supplementary Fig. 3bd). Together, our data indicates that the downregulation of YAP1 transcriptional output is an integral component of the global transcriptional response to proteotoxic stress, and it is also under nuclear speckles control.

Given the established roles of nuclear speckles in mRNA processing28, 29, 30, we next investigated whether SON also regulates mRNA splicing dynamics. While SON manipulation has no effects on the overall transcriptional state of mature mRNA under basal DMSO condition (Supplementary Fig. 4a bottom) (consistent with total mRNA shown in Fig. 1a), it exerted profound effects on the pre-mRNA level (Supplementary Fig. 4a top), indicating a change in splicing dynamics. By either estimating the relative splicing rates among different groups under basal condition using a simple first-order kinetic model of transcription (see Materials and Methods) or quantifying global intron retention events using the iRead algorithm31, we found that SON increases the splicing rates and improves the splicing fidelity of genes involved in proteostasis and RNA metabolism, and negatively regulates those involved in YAP1-related processes of cell migration, axon guidance, cell adhesion and EMT (Supplementary Figs. 4be, 5ad and 6ad). The significant enrichment in mRNA processing genes themselves under SON control is consistent with known potent autoregulation of splicing factors32, 33. Furthermore, we also found that SON can activate and repress the mature mRNA expression of a select set of proteostasis (albeit very modestly) and YAP1 target genes, respectively, under basal DMSO conditions (Supplementary Fig. 7ac). Finally, we observed that SON can also activate an anti-viral response gene signature under basal DMSO condition (Supplementary Fig. 7b), suggesting a potential broader implication of nuclear speckle rejuvenation in boosting innate immunity. Collectively, our results demonstrated that genetically rejuvenating nuclear speckles reprograms global proteostasis and YAP1 transcriptional output in an opposing manner, under both basal and proteotoxic stress conditions.

Our results thus far demonstrated a tripartite network where nuclear speckle rejuvenation by SON boosts proteostasis and suppresses YAP1. Two possible topologies of the network exist. In model one, nuclear speckles can signal both proteostasis and YAP1 signaling directly (Supplementary Fig. 8a, model 1), while in model 2, nuclear speckles repress YAP1 downstream of increased proteostasis gene program (Supplementary Fig. 8a, model 2). To distinguish between the two models, we examined a recently published RNA-seq dataset in HEK293T cells treated with DMSO, Thap or a very specific XBP1s small molecule activator IXA434, 35, 36. While IXA4 can induce a robust proteostasis gene signature similar to that of Thap, it failed to repress YAP1 transcriptional output genes as Thap did (Supplementary Fig. 8bd). Collectively these results support the first model where nuclear speckles can program proteostasis gene expression and YAP1 transcriptional output in parallel, likely via promoting physical interaction between nuclear speckles and XBP1s for the former, and triggering YAP1 nuclear exclusion for the latter (Supplementary Fig. 8e). We speculate the opposing changes in proteostasis and YAP1 signaling may reflect an energetic trade-off between proteostasis and the control of cell dynamics under proteotoxic stress (Supplementary Fig. 8f).

High-throughput screen (HTS) identified pyrvinium pamoate (PP) as a small-molecule rejuvenator of the nuclear speckle.

Having established the proof-of-principle of nuclear rejuvenation via SON OE, we next explore the feasibility of rejuvenating nuclear speckles pharmacologically, for two reasons. First, compared to the challenging implementation of SON-based gene therapy, which faces obstacles due to the considerable size (~7kb) of SON’s open reading frame, using small molecules to boost nuclear speckle activity may offer superior therapeutic potential. Secondly, we wanted to utilize an orthogonal approach to further demonstrate the opposing transcriptional changes of proteostasis and YAP1 signaling following nuclear speckle rejuvenation. Since SON OE and KD reduced and increased the sphericity of nuclear speckles19, respectively, putative nuclear speckle rejuvenators are expected to reduce the sphericity (and increase the diffuseness or irregularity) of speckles.

We started with a library of over 2500 FDA-approved drugs and ran a primary HTS on our previously described EGFP::SC35 (SRSF2) MEFs (EGFP was knocked in to the N-terminus of endogenous Srsf2 locus, which is a well-established marker for nuclear speckles9) to identify compounds that could alter nuclear speckles sphericity (Fig. 2a). As a quality control, our primary screen successfully identified four histone deacetylase inhibitors that produced much more spherical nuclear speckles approaching and/or exceeding r=0.9 (Supplementary Fig. 9a, b), in alignment with a previous study37. In the end, we identified five compounds - the tyrosine kinase inhibitors nintedanib (NB) and ponatinib (PB), the anti-microbial proflavine hemisulfate (PH) and proflavine, and the anthelmintic pyrvinium pamoate (PP) – having the ability to both decrease nuclear speckles sphericity and amplify Perk-promoter driven dGFP expression (Perk is a UPR target and exhibits 12h rhythms of expression3) in a dose-dependent manner (Fig. 2b, and Supplementary Fig. 9ce). For PP, a clear dose response in reducing the nuclear speckle sphericity was observed between 0 and 0.3μM, and further increasing its concentration failed to further decrease the sphericity (Fig. 2b and Supplementary Fig. 9c). PP also increased the perimeter of nuclear speckles (Fig. 2c), indicating that PP can increase the surface area of nuclear speckles in the three-dimensional space of the nucleus.

Fig. 2. HTS identifies pyrvinium as a SON-dependent nuclear speckle rejuvenator.

Fig. 2.

(a) Workflow detailing our initial drug screen and subsequent steps to identify compounds that affect the nuclear speckle morphology in a dose-dependent manner and amplifies the UPR. (b) Dose-dependent effect on nuclear speckles morphology by PP, with a representative image of nuclear speckles under DMSO or 0.1μM of PP (n=25~57). (c) Quantification of total area-normalized perimeter of nuclear speckles in control and 1μM PP condition per cell (n=26 for DMSO and n=35 for PP). (d) GSEA analysis showing a similar transcriptome signature between PP-upregulated genes and 461 genes further amplified by SON OE during ER stress. (e) GSEA analysis showing a similar transcriptome signature between PP-downregulated genes and 901 genes further blunted by SON OE during ER stress. (f) Volcano plot showing fold change by PP versus log transformed p values. Genes induced or repressed by at least 1.41-fold with a p value smaller than 0.05 are boxed. (g) GO analysis of differentially expressed genes by PP. (h) Representative images and quantification of sphericity of GFP signal from GFP::SRSF2 MEFs with scrambled or Son siRNA treated with DMSO or increasing concentration of PP for 25 hours (n=31~81). (i) Log2 normalized fold change in response to PP treatment (0.3 μm) for 24 hours in control and SON KD MEFs with p values shown for one tailed t-test (n=3). All data mean ± S.E.M. Statistical tests used: unpaired one-tailed Student’s t-test for c and i. Ordinary one-way ANOVA for b and h.

To determine which of these drugs are bona fide nuclear rejuvenators, we performed mRNA-Seq on MEFs treated with NB, PB, PH or PP for 24 hours and compared the transcriptome signature of each drug (Supplementary Fig. 10a, and Supplementary Table2) with those of SON OE and KD under both DMSO and Tu conditions. Gene set enrichment analysis (GSEA)38 using either those 461 Tu-induced or 901 Tu-repressed genes (depicted in Fig. 1e) revealed PP as the only drug triggering a transcriptional response with strong resemblance to SON OE cells in response to ER stress (Fig. 2d, e and Supplementary Fig. 10b). These included both upregulated genes implicated in protein quality control and downregulated genes involved in the regulation of cell dynamics (Fig. 2f, g and Supplementary Fig. 10c, d). LISA analysis on differentially expressed genes by PP revealed bZIP TFs ATF4 and YAP1 among top transcriptional regulators of upregulated and downregulated genes, respectively (Supplementary Fig. 10e). 1μm PP increased the expression of UPR and integrated stress response (ISR) TFs – XBP1s and ATF4 - at both the mRNA and protein level (Supplementary Fig. 10f, g) and induced nuclear exclusion of YAP1 (Supplementary Fig. 10h), while not affecting SON level (Supplementary Fig. 10i).

Our analysis so far suggested the PP induced a transcriptional signature that resembles a mixture of responding to SON OE and Tu treatment. We performed additional comparative transcriptome analysis to further validate this conclusion. First, when comparing the fold induction or repression of gene expression by PP and Tu, the signature of PP is more similar to that of Tu under SON OE compared to under SON KD condition (p= 0.00195 by Chow tests) (Supplementary Fig. 11a). Secondly, similar to SON OE (Supplementary Fig. 7b), PP also induced expression of genes involved in anti-viral response (Supplementary Fig. 11bf), distinct from those induced under ER stress. Thirdly, GSEA indicated a strong resemblance of gene signatures repressed by PP and SON OE that are enriched in the control of cell dynamics, under basal conditions in the absence of ER stress (Supplementary Fig. 12ae). Lastly, like SON (Supplementary Figs. 5d and 6d), PP also improves the splicing fidelity of splicing genes themselves (Supplementary Fig 13af), again reflecting autoregulation of splicing factors. Taken together, these results indicate that PP is a bona fide nuclear speckle rejuvenator that at 1μM induces a transcriptional signature in MEFs highly similar to that of SON OE cells under both basal and proteotoxic stress conditions.

PP reduces the surface tension of nuclear speckle condensates via targeting SON IDR.

To confirm that PP rejuvenates nuclear speckles in a SON-dependent manner, we knocked down Son via siRNA in MEFs. Son knockdown leads to smaller and more spherical speckles, consistent with our previous study19 (Fig. 2h). Importantly, PP’s ability to reduce nuclear speckle sphericity is abolished in Son knockdown MEFs (Fig. 2h). Subsequently, Son knockdown impaired PP’s ability to both activate protein quality control gene expression and repress YAP1 transcriptional output (Fig. 2i). To determine whether PP can physically interact with SON in MEFs, we performed cellular thermal shift assay (CETSA), which is based on ligand-induced thermal stabilization of target proteins, whereas unbound proteins denature, aggregate and precipitate at elevated temperatures, ligand-bound proteins remain soluble due to increased stability39, 40. Using a SON-specific antibody (Supplementary Fig. 14a), we found that PP induced a thermal shift of SON with a direction consistent with stabilization (Fig. 3a). As negative controls, we found that PP does not stabilize SRSF2 (SC35), another nuclear speckle protein, or the ISR/UPR TF ATF4, whose expression is nonetheless significantly increased by PP (Fig. 3b and Supplementary Fig. 14b).

Fig. 3. PP targets SON to reduce the interfacial tension of nuclear speckles.

Fig. 3.

CETSA of SON (a) and GFP::SRSF2 (b) with 3μM PP. Both representative blot and quantification from independent replicates are shown. (SON: n=6 for DMSO and n=5 for PP; GFP::SRSF2: n=3 for both DMSO and PP). (c) Computational prediction of IDR in mouse SON. (d) Diagram illustrating how surface tension influences droplets coalescence kinetics. (e, f) Representative images of droplet formation assay with different recombinant proteins (e) and quantification (f) of area-normalized perimeter changes in the time span of 20 minutes with 125mM NaCl (n=2~3). (g-i) Representative images of droplet formation with NE-supplemented SON IDR2 with increasing concentration of PP (g) and quantification of sphericity (n=12 for with NE and n=42 for without NE) (h) and area-normalized perimeter changes (n=2) (i) in the time span of 20 minutes. (j-m) Diagram of droplet formation assay where SON IDR2 is expected to compartmentalize splicing factors, including GFP::SRSF2 into the nuclear speckle-like condensates. GFP::SRSF2 is expected to exhibit a broader spatial distribution than the SON IDR2 core (j). Representative images (k) and quantification of sphericity (l) and spatial distribution of mCherry::SON IDR2 and GFP::SRSF2 (m). (n, o) Mouse genomic DNA was further added to the solution. Representative images (n) and quantification (o) of spatial distribution of mCherry::SON IDR2, GFP::SRSF2 and DNA. All data mean ± S.E.M. Statistical tests used: mixed-effects analysis for a, b and m. Ordinary one-way ANOVA for f, h, I and l. Two-way RM ANOVA for o.

SON is the central scaffold protein of nuclear speckles41, 42, 43, and its ~12h rhythmic concentration fluctuation drives ~12h rhythmic nuclear speckles LLPS dynamics and chromatin binding alternating between either a diffuse and chromatin-associated state or a punctate and chromatin-dissociated state9. By contrast, SRSF2(SC35) is one of the critical subunits of the spliceosomes, which have a broader spatial distribution also occupying the periphery of nuclear speckles, particularly at the interface between nuclear speckles and the nucleoplasm or chromatin and is not essential for speckle formation44, 45. In MEFs, PP generated a more diffuse and irregular (less spherical) nuclear speckles with larger surface area (Fig. 2b, c), suggesting that PP could influence nuclear speckle condensates material properties, likely via reducing the surface tension of speckles (surface tension is the tendency of liquid droplets to minimize the total surface area, therefore an increased surface area is suggestive of reduced surface tension46). Given the CETSA data indicating PP can bind to SON directly, we next tested whether PP can directly impact the condensates formation of two nuclear speckle protein, SRSF2 and SON, using an in vitro droplet formation assay47. Using different computational algorithms to search for intrinsically disordered region (IDR)48, 49, 50, we identified two IDRs at the N and C terminals of mouse SON, and long stretches of IDRs spanning two-thirds of mouse SRSF2 (Fig. 3c and Supplementary Fig. 14c). We separately cloned the regions encoding both SON IDRs and the full-length SRSF2 into the C-terminal of mCherry and purified recombinant proteins from E. coli (Supplementary Fig. 14d). Purified recombinant proteins were added to buffers containing 10% crowding reagents PEG-8000 as previously described47. Confocal fluorescence microscopy of the different protein solutions revealed mCherry positive, micron-sized spherical droplets freely moving in solution and wetting the surface of the glass coverslip (Supplementary Movies 1–3). All proteins droplets were highly spherical, exhibited fusion/coalescence behaviors (Supplementary Movies 1–3), and scaled in size and number positively with increasing concentration of proteins and negatively with increasing salt concentration (Supplementary Fig. 14e, f), all properties expected for liquid-like droplets47.

Due to surface tension, small droplets will eventually morph into a fewer number of large droplets, resulting in a net decrease of surface area, either via coalescence or Ostwald ripening51 (Fig. 3d), which was seen for all protein droplets after 20 minutes of time lapse imaging (Fig. 3e, f, and Supplementary Fig. 14g). Addition of PP to SON IDR2, but not SON IDR1 and SRSF2 protein solutions, significantly reduced the kinetics of this process in a dose-dependent manner, resulting in a negligible decrease of relative surface area after 20 minutes (Fig. 3df, and Supplementary Fig. 14g, h). The significance of SON IDR2 is reinforced by the substantial evolutionary conservation of its sequences across seven distinct species spanning wide phylogenetic distances from zebra fish to humans (Supplementary Fig. 14i).

To better recapitulate the complex compositions of nuclear speckles in the cell-free system, we further supplemented recombinant mCherry-SON IDR2 with nuclear extract (NE) from Hela cells that include all active components of transcription and splicing factors (Supplementary Fig. 15a)47. Mass spectrometry confirmed that Hela NE-supplemented mCherry-SON IDR2 condensates preferentially compartmentalized splicing factors (including SRSF2), with twelve of the top fifteen enriched proteins previously identified in nuclear speckles52, 53 (Supplementary Fig. 15b, c). By contrast, other nuclear proteins like proteasome subunits, DNA repair factors or general transcription factors were not enriched in the reconstituted condensates (Supplementary Fig. 15b, c). These condensates further exhibited less spherical morphology, had increased number and total size (Supplementary Fig. 15d, e), features expected from nuclear speckle-like condensates with heterogeneous viscoelastic properties54. Importantly, the addition of PP reduced both the sphericity and surface tension of Hela NE-supplemented SON IDR2 condensates in a dose-dependent manner (Fig. 3gi), consistent with the observed effects of PP on nuclear speckle morphology in cells. Together, these data suggest that PP can reduce the surface tension to stabilize both homotypic and heterotypical NE-supplemented SON condensates in a cell-free system, via interacting with SON C-terminal IDR2.

To investigate whether PP may affect the relative spatial distribution of SON and SRSF2 within nuclear speckles, we further supplemented recombinant mCherry-SON IDR2 with NE from GFP::SRSF2-expressing MEFs (Fig. 3j). The resulting nuclear speckle-like condensates recapitulated the anticipated spatial distribution of SON and SRSF2 proteins, with the former located at the center, and the latter exhibiting a broader distribution with its highest concentration often observed at 350nm away from the SON IDR2 center (Supplementary Fig. 15f and Supplementary Movie 4). While PP does not alter the relative spatial distribution of SON IDR2 and SRSF2, it reduced their sphericity, and markedly increased SRSF2 content at the periphery of nuclear speckle-like condensates (Fig. 3km). To determine whether PP may also influence the wetting of genomic DNA by nuclear speckles55, we further added mouse genomic DNA into the droplet solution. In accordance with observations in intact cells, nuclear speckle condensates largely don’t mix with but can wet the DNA (Supplementary Fig. 15g). Interestingly, the addition of PP more than doubled the wetting of genomic DNA by the reconstituted nuclear speckles (Fig. 3n, o).

To validate that PP can alter the nuclear speckle LLPS properties in vitro in the context of intact cells, we further performed 1,6 hexanediol sensitivity assay56 using the same EGFP::SRSF2 MEFs9. A short term 1,6 hexanediol treatment preferentially dissolves liquid but not solid condensates, thus a change in the sensitivity to 1,6 hexanediol reflects an alteration in the LLPS property of a given condensates56. As demonstrated in Supplementary Fig. 16a, b, PP desensitized SRSF2 to the increasing concentrations of 1,6 hexanediol. This effect was not observed on two other biomolecular condensates, the nuclear MED147 and cytosolic GW182 present in P-bodies57 (Supplementary Fig. 16c, d). Taken together, these results suggest a mechanism where PP can reduce the surface/interfacial tension of nuclear speckles via targeting SON IDR, leading to larger surface areas with increased SRSF2 content at the periphery, increased wetting of genomic DNA and subsequently a higher portion of spliceosomes stably engaging in active RNA processing and transcription elongation of proteostasis genes. Since no active transcription occurs in the in vitro droplet formation assay, these results demonstrate that PP-mediated nuclear speckle LLPS change is a cause, rather than a consequence of or response to, global transcriptional reprogramming.

PP reduces both pathological Tau and rhodopsin levels by boosting ALP and UPS, at the expense of YAP1 signaling.

Since PP treatment leads to a global increase of protein quality control gene expression, we went on to determine the effects of PP on global protein synthesis, and degradation via UPS and ALP in MEFs under two different concentrations: 0.1 and 1μM. Using puromycin incorporation assay58, we found that 0.1 μM of PP did not alter global protein synthesis (Fig. 4a, b). The lack of effects of 0.1 μM of PP on global translation was further confirmed by the lack of changes of p-eIF2α and ATF4 level (Supplementary Fig. 17a, b). To quantify the UPS activity, we treated MEFs with PP alone or in combination with the proteasome inhibitor MG132 and blotted for high molecular weight poly-ubiquitinated proteins. 0.1 μM PP led to a significant reduction of poly-ubiquitinated protein levels (Fig. 4c, d), and co-treatment with MG132 restored the level of poly-ubiquitinated proteins to a level similar to that of MG132 treatment alone (Fig. 4c, d). We further directly measured the activity of 20S proteasome core and found 0.1 μM PP significantly increased the 20S proteasome activity in a SON-dependent manner (Supplementary Fig. 17c). These data collectively indicated that 0.1 μM PP increases UPS-mediated degradation of poly-ubiquitinated protein. To quantify the autophagic flux, we utilized a tandem LC3 reporter mCherry-GFP-LC3 where an increase in the number of red-fluorescent cytosolic puncta indicates increased autolysosome formation and autophagic flux (Fig. 4e)59. 0.1 μM PP markedly increased the formation of autolysosomes (Fig. 4f). To verify this result, we further blocked autophagy at the late stage autophagosome-lysosome fusion step using Bafilomycin A1 (BafA)60, and quantified the level of LC3I and LC3II with or without PP. 0.1 μM PP treatment resulted in an increased ratio of LC3II/I and reduced level of LC3II, both of which were significantly increased by BafA co-treatment to a level similar or higher than what was observed under DMSO condition (Supplementary Fig. 17d,e). These results collectively indicate that 0.1 μM PP also augments autophagic flux. Compared to 0.1μM PP, 1μM PP leads to a global translation repression (Supplementary Fig. 18a, b) concomitant with ATF4 induction (Supplementary Fig. 10g), but is still able to promote both UPS and ALP (Supplementary Fig. 18cf). In sum, these findings demonstrate that nanomolar concentrations of PP effectively rejuvenate nuclear speckles, enhancing both the UPS and ALP without inducing cellular stress. However, at higher micromolar concentrations, PP not only acts as a nuclear speckle rejuvenator but also induces cellular stress, likely due to its known inhibitory effects on mitochondrial activity61, 62.

Fig. 4. PP reduces pathological Tau and Rhodopsin level by boosting autophagy and UPS activity.

Fig. 4.

(a-d) MEFs were treated with DMSO or 0.1μM PP for ~24 hours and then co-treated with or without puromycin (10 μg/mL for 30 minutes), or MG132 (10μM for 110 minutes) (n=4 for all samples). Western blot and quantification of puromycin-incorporated proteins (a, b), and poly-ubiquitinated protein (c, d). (e, f) MEFs were treated with DMSO or 0.1μM PP for ~24 hours and autophagic flux was measured via a mCherry::GFP::LC3 fusion protein reporter. Chimeric proteins comprising LC3B fused with both GFP and mCherry offer a method to track autophagic flux. Autophagosomes (APG) marked by mCherry::GFP::LC3 exhibit both mCherry and GFP signals. Following fusion with lysosome to form autolysosome (AL), GFP signals diminish significantly in the acidic environment, while mCherry signals remain relatively stable. (e). Quantification of the number of APG, AL and total vesicles (f). n=23 for DMSO and 29 for PP. (g, h) NIH3T3 RHOP23H cells were treated with 0.1μM PP for 24 hours and Western blot (g) and quantification (h) of RHOP23H level (n=3). (i, j) 0.1μM PP-treated NIH3T3 RHOP23H cells were co-treated with or without 1μM SBI-0206965 for 24 hours. Western blot (i) and quantification (j) of RHOP23H level (n=3). (k-m) Tau (P301S)-expressing primary neurons were treated with increasing concentration of PP for 24 hours, and western blot and quantification of different proteins (n=4 for HT7, PHF1 and LC3, and n=6 for P62) (k) or treated with 0.1μM PP for 12h hours in the presence or absence of BafA (50nM) in the last hour and western blot (l) and quantification (n=4 for total Tau and P62 and n=8 for p-Tau) (m) of different proteins. (n) Tau P301S-expressing neurons were treated with DMSO or 0.1μM PP for 12 hours and qPCR of selective proteostasis and YAP1 target genes (n=6~11). (o) MEFs were treated with DMSO, PP (1μM), XMU-MP-1 (1μM) or XMU-MP-1+PP for 24 hours, and qPCR of protein quality control and YAP1s output gene expression quantified as log transformed fold change under DMSO or XMU-MP-1 condition by PP (n=3). All data mean ± S.E.M. Statistical tests used: unpaired one-tailed Student’s t-test for b, c, f, h, j, m, n and o. Ordinary one-way ANOVA for k.

Decline of both ALP and UPS are associated with proteinopathies such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD) and a subtype of Retinitis pigmentosa (RP) with RHODOPSIN (RHO) mutations63. To determine whether PP can protect against proteinopathies via boosting UPS and ALP, we focused on two different diseases, a genetic form of RP with a mutant RHO, and tauopathy common in both AD and FTD. RP causes blindness via the primary loss of rod cells and secondary loss of cone cells. Proline to histidine at codon 23 (P23H) is the most common mutation in RHO protein, resulting in autosomal dominant RP in humans64. The heterozygous RhoP23H/+ knock-in mouse develops progressive retinal degeneration that resembles the RP phenotype in patients65, 66. Recently, several studies suggested that boosting ERAD can protect against mouse models of RP by increasing elimination of the mutant RHOP23H protein65, 67, 68, 69. To determine if PP can also reduce RHOP23H level, we used a NIH3T3 cell line ectopically expressing RHOP23H protein70. Treating this cell line with 0.1 μM of PP for 24 hours led to a reduction of both monomer and dimer forms of RHOP23H protein in a SON-dependent manner (Fig. 4g, h, and Supplementary Fig. 19a, b). Blocking autophagy with ULK1/2 inhibitor SBI-0206965 or BafA, and UPS with MG132, respectively, abolished the effect of PP on reducing RHOP23H protein level (Fig. 4i, j, and Supplementary Fig. 19cf), suggesting that both increased ALP and UPS are responsible for the increased elimination of RHOP23H by PP.

Both UPS and ALP are also involved in the degradation of tau protein in tauopathies71, 72, 73, 74. To test the effect of PP on tau proteostasis in mouse primary neuronal cultures, we overexpressed human Tau carrying P301S mutation - an FTD-causing mutation in the human MAPT gene (Tau)75. After treatment with increasing concentrations of PP for 24 hours, a decline in both total and phosphorylated Tau (Ser396/404) was observed in a dose-dependent manner, with approximately 50% and 65% reduction in total and p-Tau, respectively, detected at 100nM PP (Fig. 4k). It is noteworthy that these reductions in Tau level occurred without any observable signs of cellular toxicity (Supplementary Fig. 19g). PP also promoted autophagic flux in neurons, as demonstrated by an increase of LC3 II/I ratio, decreased level of p62/SQSTM1 and increased autolysosome formation (Fig. 4k and Supplementary Fig. 19h, i)76. Blocking autophagic flux with BafA dampened the effects of PP on reducing cellular Tau level (Fig. 4l, m). As observed in fibroblasts, PP also promoted UPS activity in neurons; however, inhibiting proteasome activity with MG132 has minimal effects on PP’s ability to reduce Tau level (Supplementary Fig. 19j, k). Consistent with PP’s ability to boost UPS and ALP, PP increased the expression of genes involved in both pathways in P301S hTau-expressing neurons (Fig. 4n). Collectively, these results indicate that increased autophagy flux largely underlies PP’s effect in reducing Tau burden in mouse primary neurons.

Nuclear speckle rejuvenation by SON OE or PP increases global protein quality control at the cost of reduced YAP1 signaling in both MEFs (Fig. 2i) and neurons (Fig. 4n), raising an interesting question of whether the downregulation of YAP1 signaling also contributed to PP’s efficacy in alleviating proteinopathy. To address this question, we restored YAP1 signaling with previously published YAP1 activators XMU-MP-1 and/or TRULI77, 78,79. We found that while XMU-MP-1 antagonized the downregulation of YAP1 target genes by PP as expected, it also potently dampened the upregulation of proteostasis genes (Fig. 4o). In addition, both XMU-MP-1 and TRULI negated PP’s effect on reducing RHOP23H level in NIH3T3 cells (Supplementary Fig. 20ad). In addition, knocking down MST1, a kinase that inhibits YAP1 activity80, similarly blocked the ability of PP to reduce RHOP23H level (Supplementary Fig. 20e, f). Similarly, TRULI also blocked PP’s effect on reducing Tau level in primary neurons (Supplementary Fig. 20g, h). These results suggest that for nuclear speckle rejuvenation to achieve the maximum effectiveness in alleviating proteinopathy, it is essential to simultaneously uphold heightened protein quality control and reduced YAP1 activity.

PP protects against mouse retina degeneration ex vivo and alleviates tauopathy in Drosophila.

Next, we assessed the effectiveness of PP in ameliorating proteinopathies by utilizing animal models of RP and tauopathy. To determine whether PP has the potential to restore gene expression changes in the retina of RhoP23H/+ mice, we performed RNA-seq in the retina of one and three months old wild-type and RhoP23H/+ mice and compared the gene signatures of RhoP23H/+ retina with that of PP (Fig. 5ae). In the retina of one month-old RhoP23H/+ mice, we observed a significant downregulation of protein transport and autophagy gene expression that showed large convergence with those upregulated by PP (Fig. 5a, b). This includes Reep6 gene, which regulates protein trafficking in the ER and its loss-of-function mutation causes autosomal-recessive RP in both humans and mice (Fig. 5e)81. By three months, the downregulation of proteostasis gene expression persists in RhoP23H/+ mice retina, concomitant with a significant increase of YAP1-mediated cell dynamics gene expression that also overlaps with PP-downregulated genes (Fig. 5ce). To directly test the efficacy of PP in protecting against RP, we treated retina explants isolated from RhoP23H/+ mice with nanomolar range of PP for 10 days, and visible light optical coherence tomography (vis-OCT) imaging82 revealed a remarkable efficacy of PP in safeguarding the mouse RhoP23H/+ retina explants from degeneration. Notably, the cell counts in the outer nuclear layer closely resembled that of the WT retina explant control (Fig. 5fj), indicating the protective potential of PP against degenerative processes. Moreover, no noticeable indications of toxicity were observed throughout the entire duration of the experiment, further highlighting the safety of PP as a promising therapeutic candidate.

Fig. 5. PP alleviates proteinopathies in preclinical models.

Fig. 5.

(a-e) RNA-seq was performed in the retina of 1 and 3 months-old wild-type and Rho P23H/+ mice. GO of differentially expressed genes (DEG) (FDR<0.1) (a, c) and GSEA comparing these DEG with that of PP (b, d). Heatmap of selective genes (e). (f-j) Retina explants isolated either from RhoP23H/+ mice P15 and cultured with PP or DMSO vehicle control or from wild-type mice cultured for 10 days ex vivo. (f) The morphology retinae were imaged and scanned before (Day 0) and after treatment (Day 10) by a webcam (top) and visible light optical coherence tomography (vis-OCT) with tissue thickness shown as a heatmap with a color legend indicating thickness from 0–300 μm (bottom). Scale, 1 mm. (g) and (h) are bar plots of retinal thickness and volume, respectively, measured from the vis-OCT scanning data. n=4~5. (i) Representative retinal histology images of the retina explants cultured for 10 days with black bars showing the outer nuclear layer (ONL). (j) The nuclei count in the outer nuclei layer (ONL) along six horizontal positions at peripheral-central-peripheral positions across each cross-section in (i). n=3–4. (k-n) Male C155>UAS-hTau1.13 and C155 flies were fed with either standard diet or diet supplemented with 25μM PP. Quantification of distance travelled from larval crawling assay (n=10) (k), climbing index score from adult fly climbing assay at 14 days and 21 days (n=5) (l) of age, and lifespan assay (m) (n=32 for C155-Ctrl, n=65 for hTau-Ctrl and n=44 for hTau-PP). (n) Western blot and quantification of p-Tau (AT8 antibody) and total Tau (DAKO antibody) level in 21 days C155>UAS-hTau1.13 flies fed with control or PP diet n=4. All data mean ± S.E.M. Statistical tests used: two-way ANOVA and Turkey multiple comparison for g and h, two-way ANOVA for j, unpaired one-tailed Student’s t-test for k, l (day 21) and n. Log-rank (Mantel-Cox) test for m.

Pan-neuronal expression of wildtype human MAPT gene in Drosophila recapitulates essential features of tauopathies, including hyperphosphorylated and misfolded tau, age-dependent neuron loss, and reduced life span83, and SON IDR2 sequence is also conserved in flies (Supplementary Fig 14i). Thus, we next tested the efficacy of PP in ameliorating tauopathy in male flies that express 2N4R isoform of human Tau (MAPT) pan-neuronally [elavc155-Gal4: UAS-hTau1.13 (C155>UAS-hTau1.13)] as well as in control elav c155-Gal4 (C155) flies84. Both C155>UAS-hTau1.13 and C155 flies were fed with either standard diet or diet supplemented with 25μM PP, which did not affect the normal development and growth of flies despite its effect in attenuating WNT and YAP1 signaling85. We quantified disease progression at different stages of fly development with both larval crawling and adult fly climbing assay at 14 and 21 days of age. PP feeding preserved motor function in C155>UAS-hTau1.13 third instar larvae and adult flies, with their locomotor performance restored to a level similar to or even slightly higher than control C155 flies fed with a standard diet (Fig. 5k, l). Notably, PP also enhanced the motor function of adult wild-type control (C155) flies at 21 days of age (Fig. 5l). This improvement is likely linked to PP’s ability to promote overall proteostasis, particularly protein turnover rates, a process known to prolong health and lifespan in flies86. PP further extended the median lifespan of C155>UAS-hTau1.13 flies by 16% from 51 to 59 days (Fig. 5m). Consistent with the overall phenotypes, PP significantly reduced the level of p-Tau and to a lesser extent total Tau in the brains of 21 days-old C155>UAS-hTau1.13 flies (Fig. 5n). Since p-Tau are prone to misfolding and aggregation, it supports the notion that PP is increasing the overall capacity of protein quality control to remove misfolded and aggregated proteins, while having negligible effects on normal protein functions.

PP has the potential for treating tauopathy in humans.

To determine the potential of PP for treating tauopathy in humans, we studied whether gene expression signatures that are opposite of PP can be observed in brain regions of human subjects with AD. We initially performed a post-hoc analysis of a total of nineteen bulk RNA-seq datasets encompassing hippocampus, entorhinal cortex, temporal cortex and frontal cortex regions in control and AD human subjects87, and found that genes repressed by PP have increased expression in all four brain regions of human subjects with AD (such as YAP1, TEAD1 and AMOT) (Supplementary Figs. 21 and 22a). By contrast, genes that were upregulated by PP displayed significantly decreased expression in temporal cortex (such as genes involved in ERAD: EDEM1, SEL1L, autophagy: ATG13, protein folding: HYOU1, and tRNA aminoacylation: GARS, IARS) (Supplementary Figs. 21 and 22a). To validate these findings, we further analyzed an independent single-nucleus RNA-seq (snRNA-Seq) dataset in the prefrontal cortex regions of human individuals with varying degrees of AD pathology (Supplementary Fig. 22b)88. We found that proteostasis genes upregulated by PP are consistently downregulated in all cell types with strong prominence in neurons and oligodendrocytes in both early and late-stage human AD subjects (Supplementary Fig. 22c, d). Genes that are downregulated by PP (those enriched in regulation of cell dynamics by YAP1) are initially downregulated in all cell types in the early stage but significantly upregulated during the late stage of AD in all cell types but inhibitory neurons (Supplementary Fig. 22e, f). This early to late AD progression is concomitant with strong increase of tauopathy, but not the amyloid burden in these individuals (Supplementary Fig. 22b). Consistent with in vivo data, we also observed a significant decrease of proteostasis gene expression as well as an increase of YAP1-TEAD2 target gene expression in human induced pluripotent stem cells (iPSC)-derived neurons that express the P301S 4R-Tau when compared to wild-type 4R-Tau control cells (Fig. 6ac)74. These upregulated YAP1-TEAD2 target genes also overlap with those repressed by PP (Fig. 6b).

Fig. 6. PP rejuvenates nuclear speckles and alleviates tau burden in human iPSC-neurons expressing mutant Tau.

Fig. 6.

(a) GO analysis of up and downregulated genes in 4R-Tau P301S iPSC neurons reported in74. (b) GSEA comparing genes upregulated in 4R-Tau P301S iPSC neurons with those downregulated by PP. (c) Motif analysis of promoters of genes upregulated in 4R-Tau P301S iPSC neurons compared to 4R-Tau (FDR<0.05) revealed top enriched motif of TEAD2. (d, e) Wild-type and V337M Tau-expressing iPSC-neurons were treated with DMSO or PP (10nM) for 12 hours, and IF against nuclear speckle (Ab11826 against SRRM2), p-Tau (Ser422) and chromatin (DAPI) were performed. Representative images (d) and quantification of nuclear speckle sphericity and area, intensity of nuclear, cytosol and total level of p-Tau (Ser422) and ratio of cytosol versus nuclear level of p-Tau (e). (f, g) V337M Tau-expressing iPSC-neurons were treated with DMSO or increasing concentrations of PP for 12 hours. Representative western blot (f) and quantification (g) of total and p-Tau (Ser202/Thr205) (n=3). (h) V337M Tau-expressing iPSC-neurons were treated with DMSO or 100nM PP for 12 hours, and autophagy flux was quantified by the autophagy reporter. (i-k) Wild-type and V337M Tau-expressing iPSC-neurons were infected with scrambled shRNA or SON shRNA-encoding lentivirus and treated with DMSO or PP (100nM) for 12 hours, and IF against nuclear speckle (Ab11826 against SRRM2), p-Tau (Ser422) and chromatin (DAPI) were performed. Quantification of nuclear speckle area (i) sphericity (j), intensity of total level of p-Tau (Ser422) (k) were shown. Data: Mean ± SEM.

Finally, to directly test the efficacy of PP in reducing tauopathy in human, we utilized human iPSC-neurons harboring homozygous FTD-causing MAPT V337M mutation (herein referred to as V337M) and isogenic wild-type control cells89 and treated both cell lines with nanomolar range of PP for 12~24 hours. After 24 hours of treatment with 500 nM PP, iPSC neurons exhibited no signs of toxicity, as confirmed by the LDH release assay (Supplementary Fig. 23a, b). Moreover, the nanomolar PP treatment did not trigger cellular stress, as indicated by the unchanged p-eIF2α levels (Supplementary Fig. 23c). Immunofluorescence against nuclear speckles marker SRRM2 revealed that compared to controls, V337M iPSC-neurons exhibited aberrant nuclear speckle morphology characterized by smaller size and more spherical shape, and 12 hours of 100nM PP treatment fully restored nuclear speckle morphology to normal size and diffuseness (Fig. 6d, e). Consequently, PP markedly reduced the level of V337M p-Tau, concomitant with increased autophagic flux (Fig. 6dh). To confirm that PP rejuvenates nuclear speckles and reduces V337M p-Tau in a SON-dependent manner, we knocked down SON using lentiviral shRNA and repeated the experiment. As demonstrated in Fig. 6ik and Supplementary Fig. 23d, PP failed to rejuvenate nuclear speckles or reduce p-Tau level in the presence of SON knockdown in either wild-type or V337M Tau-expressing iPSC neurons. Together, these findings indicate that PP treatment has great potential to normalize gene expression patterns and reduce Tau burden in AD/ADRD-affected humans with severe tauopathy. Further, these results provide strong support for the decline of nuclear speckles LLPS dynamics (becoming smaller and more spherical) as a driver of tauopathy.

Discussion

Several recent studies indicate that both the decline of nuclear speckle functions and dysregulated mRNA splicing are associated with proteinopathies in humans, including tauopathy, RP and amyotrophic lateral sclerosis (ALS)90, 91, 92, 93. For example, two studies showed that elevated Tau aggregates have the capability to relocate to the nucleus, thereby directly modifying the characteristics of nuclear speckles92, 94. In agreement with this study, a recently published Tau interactome in human iPSC neurons95 also revealed strong enrichment of Tau-interacting proteins involved in regulating RNA splicing and RNA stability. Additionally, it was previously shown that cryptic splicing errors are associated with neurofibrillary tangle burden in human AD subjects96. In humans, among the 12 autosomal dominant RP genes identified, four encode ubiquitously expressed proteins involved in pre-mRNA splicing (including PRPF31, PRPF8, PRPF3 and RP9), demonstrating the important roles of RNA processing in the pathogenesis of retinal degeneration93. These studies thus provide the rationale for nuclear speckle rejuvenation as a strategy for counteracting various proteinopathies.

Exploring the therapeutic potential of targeting biomolecule condensates represents an exciting avenue for research and drug development97, 98. Our study is proof of principle demonstrating that nuclear speckle LLPS can also be therapeutically targeted. Manipulating nuclear speckle LLPS through SON overexpression is a conceptually viable approach. However, the practical implementation of this strategy presents significant challenges due to the large size of the human SON open reading frame, making it technically difficult to design gene therapy targeting SON. That said, we cannot rule out the possibility that overexpression of specific truncated SON domains, such as SON IDR2, may be sufficient to rejuvenate nuclear speckles, and future efforts will be directed toward exploring such possibilities.

Through a high-throughput drug screen, we identified PP as a small nuclear speckle rejuvenator by directly interacting with SON and modulating nuclear speckle LLPS dynamics. Interestingly, pyrvinium is enriched with cationic amines and aromatic motifs, chemical features that were predicted to partition into various nuclear condensates99. PP was originally developed as an anthelmintic drug effective for treating pinworm infections100. Moreover, it has gained strong recent interest as an anti-cancer reagent due to its ability to inhibit WNT signaling100. Our current study further expands its therapeutic values to proteinopathies, including both reducing tauopathy in neurons and flies and protecting against retina degeneration in an ex vivo mouse RP model. Future efforts should be directed toward testing the toxicity and efficacy of this drug in mouse models of neurodegenerative diseases.

While we don’t yet know the full detailed mechanisms by which PP modulates nuclear speckles dynamics and boosts proteostasis gene transcription, several lines of evidence suggest that it does so in part by reducing the surface tension and consequently increasing the surface areas of nuclear speckles via an SON-dependent manner. Our in vitro reconstitution system further showed that reduced nuclear speckles surface tension by PP further facilitates nuclear speckles wetting of chromatin46. Thus, since spliceosomes reside at the interfacial boundary between nuclear speckles and nucleoplasm/chromatin44, larger surface areas also entails a higher probability of spliceosome stably engaging in mRNA processing and transcription elongation. The mechanism by which PP reduces the surface tension of nuclear speckles is not yet fully understood. While the straightforward explanation would be that PP acts as a surfactant, this seems unlikely due to its lack of hydrophilic moieties. Alternatively, it is plausible that by interacting with SON, PP weakens the intermolecular attractive interactions among different SON proteins, and/or SON and IDRs of other nuclear speckle proteins due to screening effects, leading to an overall reduction of surface tension of nuclear speckles, similar to what is previously described for the effect of increasing salt concentration on reducing surface tension for protein condensates101, 102. The positive charge carried by pyrvinium adds to the allure of this hypothesis. Further research is needed to elucidate the precise mechanisms by which PP influences the surface tension of nuclear speckles.

Genetic and pharmacological rejuvenation of nuclear speckles by SON and pyrvinium share similar transcriptome signatures, including upregulation of extensive protein quality control gene expression, and intriguingly, downregulation of YAP1-regulated genes involved in cell migration, cell proliferation, would healing, and extracellular matrix organization. The contrasting changes in proteostasis and YAP1-mediated cell dynamics gene expression are observed in various cell lines under proteostatic stress. Consequently, both SON overexpression and ER stress inhibits YAP/TEAD transcriptional activity and impedes cell migration. These findings demonstrate that YAP1 signaling is an inherent component of global transcriptional control of proteostasis. One possible explanation for this phenomenon is that cells need to allocate their energy towards enhancing overall proteostasis, which may come at the cost of cell proliferation, migration, and extracellular matrix organization. Therefore, an energetic trade-off between proteostasis and cell dynamics control could be crucial for maintaining cellular functions when faced with fluctuating environments, such as proteotoxic stress.

Nuclear speckles play a vital role in coordinating the opposing changes observed in proteostasis and cell dynamics regulation. Elevated SON expression facilitates increased physical interactions between nuclear speckles and XBP1s, leading to augmented transcription of proteostasis genes. Since the number of proteostasis genes under the control of SON surpasses those directly regulated by XBP1s, we postulate that nuclear speckles can be recruited to additional proteostasis bZIP TFs upon rejuvenation, possibly via increased wetting between nuclear speckle and TF-mediated condensates46. Future work with unbiased profiling of nuclear speckle composition (via proximity labeling for example) can unveil the detailed molecular mechanisms through which nuclear speckle rejuvenation globally activates the proteostasis gene program. On the other hand, much less is clear on how nuclear speckle rejuvenation represses YAP1 transcription activity. Upon SON overexpression and pyrvinium treatment, we found a significantly reduced level of nuclear YAP1 protein and a lower nucleus/cytosol ratio, indicating an active nuclear exclusion of YAP1 protein. Like nuclear speckles, YAP1 can also form biomolecular condensates, and a recent study reported that YAP1 nuclear condensates and the nuclear speckles showed limited nuclear co-localization103, suggesting a low level of wetting of these two condensates under normal physiological conditions. Pending further investigation, we speculate herein that nuclear speckles rejuvenation may further reduce the wetting of these two condensates, resulting in the alteration of YAP condensate composition, and ultimately its nuclear exclusion.

While both proteostasis and YAP1 signaling are downstream of nuclear speckles, direct antagonistic reciprocal interactions between these two are likely to be present as well. A recent study reported that in Drosophila, the proteostasis output gene Bip can sequester the fly YAP1 ortholog Yorkie, in the cytoplasm to restrict Yorkie transcription output104. Conversely, in undifferentiated pleomorphic sarcoma, YAP1 can suppress PERK and ATF6-mediated UPR target expression, and treatment with the YAP1 inhibitor Verteporfin upregulated the UPR and autophagy105. The latter is further consistent with our findings showing that restoring YAP1 activity dampened the efficacy of PP on activating protein quality control gene expression and reducing proteinopathies. These findings indicate that in order to maximize the effectiveness of nuclear speckle rejuvenation, it is crucial to maintain elevated levels of protein quality control while simultaneously reducing YAP1 activity. Thus, a delicate balance between protein quality control and YAP1 activity appears essential for effective nuclear speckle rejuvenation. This observation may also explain why therapies merely aimed at activating protein quality control pathways often have limited efficacies. Supporting this notion, while reduced expression of genes involved in ERAD and autophagy are observed in the brains of individuals with AD, these subjects also exhibit elevated gene expression of YAP1, TEAD1, and other YAP1 target genes, consistent with previous studies87, 106. Increased YAP1 target gene expression was further observed in human iPSC tauopathy model as well as in the retina of Rho P23H/+ mice. These results collectively highlight the importance of suppressing YAP1 signaling as a potential strategy for managing both AD and RP.

In conclusion, our study makes substantial conceptual contributions to the broader proteostasis framework by incorporating nuclear speckle LLPS and YAP1 signaling as critical components. From a translational perspective, our research unveils the promising therapeutic potential of nuclear speckle rejuvenation in tackling proteinopathies, achieved by simultaneous activation of protein quality control and inhibition of YAP1 activity. Additionally, our findings underscore the significance of harnessing the 12-hour oscillator to unveil hidden principles of proteostasis control.

Materials and methods

Mice

For retinal explant studies, wildtype C57BL/6J and RhoP23H/+ knock-in mice (Jackson Laboratory Strain #017628) were euthanized by CO2 and retinae were isolated for culture. The animal studies were carried out in accordance with the National Institutes of Health guidelines and were granted formal approval by the University of Pittsburgh’s Institutional Animal Care and Use Committee (approval numbers IS00013119 and IS00023112).

Larva crawling assay

PP solubilized in DMSO were diluted directly into the fly medium at the final concentration of 25 μM and vortexed extensively to obtain homogeneous culture. Crawling assays were performed on 1.5% agarose plates made with a 2.3:1 combination of grape juice and water. A sample size of 10 to 15 larvae were selected for each genotype and assays were done using larvae in the third instar state. The larvae were first removed from vials and gently placed into a petri dish containing deionized water to allow for residual food to be washed off the body. After 15 seconds, the larvae were transferred to a petri dish containing the 1.5% agarose mixture and were given one minute to rest. They were then transferred to a second dish filled with the 1.5% agarose mixture and timed immediately for one minute, during which their crawling performance was measured. A transparent plastic lid was placed on top of the plates and the crawling path of the larvae were traced. Observations of the crawling activity were done under a light microscope. The brightness and distance of the light source above the plates were kept constant across all trials and genotypes. The crawling paths of the larvae were measured using FIJI ImageJ and the average distance traveled was taken for each genotype.

Adult fly climbing assay

Male adult Drosophila melanogaster flies at 14 and 21 days of age fed with a normal diet or diet supplemented with 25 μM PP were used for assessing climbing ability. Flies were grouped into cohorts of the same sex, pre-mated, and age-matched, with a maximum of 20 individuals per vial (usually 5–15). All flies used in each trial were hatched within a 3-day window. The evening prior to each assay, flies were gently transferred to fresh tubes to allow for grooming and access to food. To ensure consistent conditions, assays were conducted at approximately the same time of day with a consistent ambient light setting. A custom climbing vial was employed, divided into six compartments, each labeled with a number (1 to 6) to denote climbing speed. The vial was positioned against a white background to enhance visibility during photography. Flies were transferred from their housing vial to the climbing vial, which was covered with a plastic plate on top. To initiate the assay, the flies were gently tapped to the bottom of the vial and allowed 10 seconds to climb. A cell phone camera was used to capture a photograph of the vial. Care was taken to ensure the camera was level with the vial, all flies were visible, and the background was free from stains or spots. The number of flies in each compartment of the climbing vial was counted at each time point and recorded on a dedicated worksheet. Each cohort of flies underwent five consecutive trials, with approximately 1 minute of rest between each trial. The average score of each cohort was determined by dividing the total score by the total number of flies.

Fibroblast cell culture and drug treatment

MEFs and NIH 3T3 cells were cultured at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, glucose 4.5 g/L with phenol red) and supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate (Gibco), and penicillin (100 U/mL)-streptomycin (100 μg/mL) (Gibco). Methods for the manipulation of Son (transient knockdown or constitutive overexpression) and validation of changes to protein (SON) levels with regards to the mRNA-Seq data are previously described in9. For Tu treatment, 100 ng/mL Tu (in DMSO) for six hours was used unless otherwise noted. NB (HY-50904), PB (HY-12047), PH (HY-B0883), PP (HY-A0293), MG-132 (HY-13259), SBI-0206965 (HY-16966) and XMU-MP-1 (HY-100526) were purchased through MedChemExpress and BafA (1334) were purchased from Tocris. All drugs were handled per manufacturer instruction.

siRNA Transient Transfections

MEFs were transfected with 10μM of different siRNAs for 24~48 hours with Lipofectamine RNAiMAX reagents (Life technologies) per the manufacturer’s instructions. Sources of siRNA are as follows: siGENOME non-targeting siRNA pool (Dharmacon, D-001206–1305), siGENOME SMARTpool son siRNA (Dharmacon, L-059591–01-0005), siGENOME SMARTpool Stk3/Mst1 siRNA (Dharmacon, L-040440–00-0005), and siGENOME SMARTpool Stk4/Mst2 siRNA (Dharmacon, L-059385–00-0005).

Primary neuron cell culture and P301S-Tau virus infection

The cerebral cortices of 3–4 neonate mice (P0) were dissected on ice, the meninges were removed and placed in the cold dissection medium (DM), consisting of 6 mM MgCl2 (Sigma M1028–100 ml), 0.25 mM CaCl2 (Sigma C7902), 10 mM HEPEs (100X), 0.9% Glucose, 20 μM D-AP5 (Cayman, NC1368401), and 5 μM NBQX (Tocris Bioscience, 10–441-0). After dissection, the brain tissues were washed with DM 1~2 times and incubated with 13mL of DM containing papain (Worthington, LK003176) in 37°C water bath for 20 min. The suspension was shaken every 5 min. 10mL media containing 18 ml DM + 2 ml low OVO + 133 ul DNase I (dilute 10X low OVO and 150x DNase I to DM) were added into the suspension to stop the digestion in 37°C water bath for 5 min. The solution was taken off and 10 ml fresh solution was added in. Then the tissues were triturated until there were no visible chunks, and the solution was filtered through the 70 μm cell strainer. The cell solution was then centrifuged at 1000 rpm for 10 min and the supernatant was discarded. The cell pellet was gently resuspended in 20 ml B27/NBM/High glucose media, and the suspension was centrifuged at 850 rpm for 5 min. After that, the supernatant was taken off and B27/NBM (1 ml/mouse brain) was added to resuspend the cells until single cell solution. The cells were counted and plated onto the coverslips at 250k in 24-well plates for imaging or 800k in 12-well plates for qRT-PCR or Western blots. AAV-P301S hTau (Viro-vek) were infected at DIV1 at 100 MOI.

Human iPSC-derived neurons culture

Human iPSC-derived neurons were pre-differentiated and differentiated as described89. Briefly, iPSCs were pre-differentiated in Matrigel-coated plates or dishes in N2 Pre-Differentiation Medium containing the following: KnockOut DMEM/F12 as the base, 1× MEM non-essential amino acids, 1× N2 Supplement (Gibco/Thermo Fisher Scientific, cat. no. 17502–048), 10 ng/ml of NT-3 (PeproTech, cat. no. 450–03), 10 ng/ml of BDNF (PeproTech, cat. no. 450–02), 1 μg/ml of mouse laminin (Thermo Fisher Scientific, cat. no. 23017–015), 10 nM ROCK inhibitor and 2 μg/mlof doxycycline to induce expression of mNGN2. After 3 d, on the day referred to hereafter as Day 0, pre-differentiated cells were re-plated into BioCoat poly-D-lysine-coated plates or dishes (Corning, assorted cat. no.) in regular neuronal medium, which we refer to as +AO neuronal medium, containing the following: half DMEM/F12 (Gibco/Thermo Fisher Scientific, cat. no. 11320–033) and half neurobasal-A (Gibco/Thermo Fisher Scientific, cat. no. 10888–022) as the base, 1× MEM non-essential amino acids, 0.5× GlutaMAX Supplement (Gibco/Thermo Fisher Scientific, cat. no. 35050–061), 0.5× N2 Supplement, 0.5× B27 Supplement (Gibco/Thermo Fisher Scientific, cat. no. 17504–044), 10 ng/ml of NT-3, 10 ng/ml of BDNF and 1 μg/ml of mouse laminin. Neuronal medium was half-replaced every week.

Efficacy test of PP in Retina explant culture

Wild type and RhoP23H/+ mice were euthanized at P15, and retina explants were isolated and cultured as previously described107, 108. Briefly, eyeballs were enucleated and incubated in Ames solution containing 0.22 mM L-cysteine (Sigma-Aldrich) and 20 U papain (Worthington, Freehold NJ, USA) at 37 °C for 30 min. The digestion was stopped by transferring the eyes to Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal calf serum (FCS; Gibco) and penicillin &streptomycin antibiotics (1x, GenClone) at 4 °C for 5 min. The eye cup was made by gently removing the cornea, iris and lens. Each eye cup was flattened by four radio cuts and the sclera was then carefully peeled off from the retina:RPE complex. The retina:RPE explant was transferred to a trans well insert with 0.4-micron pore polycarbonate membrane (ThermoFisher) sitting on the surface of 1.5 mL of neurobasal-A plus medium (Gibco) containing 2% B27 supplement (Gibco) in a 6-well cell culture plate, and the RPE layer was facing down the transwell membrane. The retinal explants were cultured at 37 °C with 5% CO2.The medium was replaced with fresh medium containing 0.5 μM PP after 24 h, which was replaced again every 2 days until 10 days in culture (DIV). A visible light optical coherence tomography (vis-OCT) prototype109 was utilized to monitor the explants noninvasively at day 0 and day 10. Retinal layers were segmented automatically using a deep learning method and then manually corrected by a customized software to calculate the retinal thickness. Retina explants were collected at 10 DIV and processed for fixation, dehydration, paraffin embedding, cross-sections, dewaxing, rehydration and hematoxylin and eosin (H&E) staining110. H&E-stained slides were imaged by regular light microscopy with a color camera, and the number of nuclei in the outer nuclear layer (ONL) was calculated manually.

Autophagy reporter assay

To express the LC3 reporter in the neurons, the primary mouse neuronal cultures were infected with the homemade lentivirus-mCherry-GFP-LC3 for 7 days. The florescent signal from the vacuoles at different stages were acquired by confocal imaging. The mCherry-GFP-LC3 fluorescence images were acquired with a Leica TCS SP8 confocal system using 63x oil-immersion objective. 488 nm and 568 nm laser were used to excite the GFP and mCherry, respectively. Images were taken with the same confocal settings. Minor image adjustment (brightness and/or contrast) was performed in ImageJ. The GFP and mCherry signal collected were merged into one image to quantify the red, green, and yellow vacuoles for quantification of different types of vacuoles. The different colored fluorescent signal was manually counted in each cell, and each point represents the average number of the specific vacuole for one cultured cell. For autophagy reporter assay in MEFs, pCDH-EF1a-mCherry-EGFP-LC3B was a gift from Sang-Hun Lee (Addgene plasmid # 170446; http://n2t.net/addgene:170446; RRID:Addgene_170446)111 and purchased from addgene. Lentivirus was packaged from HEK293T cells as previously described19 and was used to infect MEFs with a MOI of 3 three times. The quantification was performed essentially the same way as in neurons.

TEAD luciferase reporter assay

MEFs with the CRISPRa system either overexpressing Son or serving as controls used in this assay were previously described in112. Briefly, cells were seeded at a density of 7000 cells per well in a 96-well plate with a clear bottom and white walls. Cells were then transfected using the Lipofectamine 3000 transfection kit (ThermoFisher #L3000015) for 22 hours with the 8xGTIIC-luciferase plasmid, a gift from Stefano Piccolo (Addgene plasmid # 34615; http://n2t.net/addgene:34615; RRID:Addgene_34615)113. The Dual-Glo Luciferase Assay System (Promega #E2920) was used with a SpectraMax i3x plate reader (Molecular Devices) to measure firefly luciferase signal (500ms integration time, 1mm from the plate read height).

Proteasome 20S activity assay

The proteasome 20S activity assay was performed per manufacturer’s instruction (Sigma Aldrich, MAK172). Briefly, MEFs transfected with scrambled control or Son siRNA were treated with DMSO or 100nM PP for 24 hours. After treatment, cells were washed with PBS and cultured in phenol-free DMEM. Assay reagents were added directly to the cells, and fluorescent signals were measured by a fluorescent plate reader 2 hours later at λex = 490 nm and and λem = 525 nm. The final signal was corrected by subtracting the fluorescence of the background blank (medium without cells) from the fluorescence of all test wells.

Scratch assay

Cells were grown until they were 100% confluent, ER stress was induced as previously described, and then a single scratch was performed with a pipette tip per well. Cells were imaged immediately after scratching (0hr) and then after 23hr. The Cell Profiler114 “Wound Healing” pipeline (https://cellprofiler.org/examples) was used to measure the “Percentage of Gap Filled”.

Immunoblot

Different cells were harvested and fractionated to produce cytosolic and nuclear lysates using the NE-PER kit (Thermo Fisher Scientific). For whole cell lysates, cells were lysed in RIPA buffer. Both protease and phosphatase inhibitors were included in the respective lysis buffer. ~47 μg of protein was separated on a 4%−15% gradient SDS-polyacrylamide gel (Bio-Rad) which were transferred to nitrocellulose membranes, stained with Ponceau S stain, washed, blocked with 5% non-fat milk, and incubated overnight at 4°C with the following primary antibodies: anti-α-Tubulin (Cell Signaling Technology (C.S.T.) #2144), anti-Lamin A/C (C.S.T. #4777), anti-SON (Abcam #121033 and LSBio LS-C803664), anti-YAP1 (C.S.T. #12395), anti-GFP (C.S.T. #2956), anti-Tau (Sigma-Aldrich, #A0024), anti-p-Tau (a gift from Dr. Peters Davies), anti-β-actin (C.S.T. #4970), anti-puromycin (BioLegend 381502), anti-ubiquitin (C.S.T. #58395), anti-LC3-I/II (C.S.T. #2775), anti-p62 (C.S.T. #23214), anti-ATF4 (C.S.T. #11815), anti-ATF6 (Novus 70B1413.1), anti-MST1 (C.S.T. #3682), anti-MST2 (C.S.T. #3952), anti-eIF2α (C.S.T. #5324), anti-p-eIF2α (Ser51) (C.S.T. #3597) and anti-XBP1s (BioLegend 658802). The 1D4 anti-rhodopsin antibody115 was obtained as a gift from Dr. Krzysztof Palczewski’s laboratory. Membranes were treated with the appropriate secondary antibody conjugated to horseradish peroxidase the following day and then ECL Prime Western Blotting Detection Reagent (Cytiva) was applied. A Bio-Rad ChemiDoc MP Imaging System was used to visualize the signal, and signal intensities were determined with ImageJ116. For anti-Rhodopsin western blot, the protein samples were not boiled before loading.

Cellular thermal shift assay (CETSA)

EGFP::SC35 MEFs with EGFP knocked into the N-terminal of mouse Srsf2 locus (previously described in9) were treated with either DMSO or 3μM PP for 50 minutes at 37°C. Cells were then trypsinized and resuspended in PBS with either DMSO or 3μM PP and 100 μL of the suspensions were distributed to PCR tubes for the thermal shift assay (three minutes at a range of temperatures). The temperatures used were: 42.0°C, 42.5°C, 43.9°C, 46.2°C, 49.3°C, 53.3°C, 57.9°C, 62.1°C, 65.2°C, 67.8°C, 69.2°C, and 70.0°C. After the samples were heated, they sat at 20°C for three minutes, were snap frozen in liquid nitrogen and thawed for three cycles to lyse the cells, and then spun at 20,000 × g for 20 minutes at 4°C. The supernatant was then removed, and immunoblotting was performed using anti-GFP (C.S.T. 2956), anti-ATF4 (C.S.T. #11815), and anti-SON (Lifespan Biosciences #LS-C803664–100), followed by appropriate secondary antibody. Band intensity on the blots were relative to the intensity of the 42°C band and were normalized so that this band’s (42°C) intensity was set equal to 1.

Protein purification and in vitro droplet formation assay

Regions of SON and the entire SRSF2 protein were fused to mCherry. cDNA encoding the SON-IDR N terminal (region 1), SON-IDR C-terminal (region 2), and SRSF2 were each cloned into the expression vector pET21a (+)-Histag-mCherry (Addgene plasmid # 70719) (Niederholtmeyer et al., 2015). The plasmids obtained were transformed into C3013 E. Coli (NEB C3013I). Fresh bacterial colonies were inoculated into LB media containing ampicillin and grown overnight at 37°C. Overnight cultures were diluted in 500mL of LB broth with ampicillin and grown at 37°C until reaching OD 0.6. IPTG was then added to 2mM, and growth continued for 3h at 37°C. The cells were pelleted and stored frozen at −80°C. Bacterial pellets were resuspended in 15mL of Buffer A (50 mM Tris-HCl, 500 mM NaCl) containing protease inhibitors (Pierce, A32965) and 10mM imidazole. The suspension was sonicated on ice for 15 cycles of 30 sec on, 30 sec off. The lysate was centrifuged for 40 minutes at 15,000 RPM at 4°C to clear debris, then added to 2mL of preequilibrated Ni-NTA agarose beads (Qiagen cat no. 30210). The agarose lysate slurry incubated for 1.5hrs at 4°C while rocking, then allowed to flow through the column. The packed agarose was washed with 15mL of Buffer A with 10mM imidazole. Protein was eluted with 5mL of Buffer A containing 15mM imidazole, 10mL Buffer A containing 100mM imidazole, then 10mL Buffer A containing 200mM imidazole. All elutions were collected in 1mL fractions. Aliquots of the collected fractions were run on an SDS-PAGE gel and stained with Imperial Protein Stain to verify the amount and purity of the protein. Fractions containing protein were combined and dialyzed against Dialysis Buffer (50mM Tris-HCl, 500mM NaCl, 10% glycerol, 1mM DTT). Recombinant mCherry fusion proteins were concentrated and desalted to 200uM protein concentration and 125mM NaCl using Amicon Ultra centrifugal filters (MilliporeSigma cat no. UFC801024) following manufacturer’s instructions. 20uM of recombinant protein was added to Droplet Buffer (50mM Tris-HCl, 10% glycerol. 1mM DTT, 10% PEG) containing indicated final salt and pyrvinium concentrations. For droplet formation assay with Hela nuclear extract supplementation, NE was added to different concentration of SON IDR2 at the final concentration of 1.5mg/ml in Droplet buffer (20mM HEPES, pH 7.9, 20% glycerol, 125mM KCl, 0.2mM EDTA, 0.5mM DTT, 10% DEG). For droplet formation assay with GFP::SRSF2 MEF nuclear extract supplementation, NE was added to 10uM SON IDR2 at the final concentration of 0.6mg/ml in Droplet buffer. A custom imaging chamber was created by placing strips of tape on a glass coverslip, forming a square. The protein solution was immediately loaded onto the center of the square and covered with a second glass coverslip. Slides were then imaged with a Leica confocal microscope with a 63x oil objective. The image series were taken over a 20-minute time span with 1 image every 30 seconds.

Mass spectrometry to profile condensates composition

Hela nuclear extract samples were thawed at room temperature, vortexed for 10 minutes, bath sonicated for 5 minutes and centrifuged at 13000g for 10 minutes at room temperature prior to quantification of total protein by a Pierce 660 Protein Assay (Thermo Scientific #22660). Condensates were obtained by centrifuging at 10,000xg for 10 minutes and resuspended in 5%SDS in 50 mM TEAB prior to total protein quantification. Protein digestion was carried out on 10 μg of protein from each sample on S-trap micro columns (Protifi) according to the manufacturer’s protocol. Following digestion, peptide samples were then dried in a speedvac and resuspended in a solution of 3% acetonitrile and 0.1% TFA and desalted using Pierce Peptide Desalting Spin Columns (Thermo Scientific # 89851). Eluants were dried in a speedvac and resusupended in a solution of 3% acetonitrile and 0.1% formic acid to a final concentration of 0.5 μg/μL. Mass spectrometry analysis was conducted on a Thermo Fisher QE-HFX coupled to a Vanquish Neo UHPLC. Approximately 1 μg of each sample was loaded onto an EASY-Spray PepMap RSLC C18 column (2 μm, 100A, 75μm × 50 cm) and eluted at 300 nl/min over a 120-minute gradient. MS1 spectra were collected at 120,000 resolution with a full scan range of 350 – 1400 m/z, a maximum injection time of 50ms and the automatic gain control (AGC) set to 3e6. The precursor selection window was 1.4 m/z and fragmentation were carried out with HCD at 28% NCE. MS2 were collected with a resolution of 30,000, a maximum injection time of 50ms and the AGC set to 1e5 and the dynamic exclusion time set to 90s. The collected MS data were analyzed using MSFragger V4.0[1] and searched against the human SwissProt database. The search parameters were set as follows: strict trypsin digestion, missing cleavage up to 2, carbamidomethylation of cysteine as static modification, oxidization of methionine and protein N terminal acetylation as variable modification, a maximal mass tolerance of 20 ppm for the precursor ions and 20ppm for the fragment ions, and false detection rate (FDR) was set to be 1%.

1,6-hexanediol treatment to examine effects of PP on LLPS

A 10% (w/v) 1,6-hexanediol (1,6-HD, MilliporeSigma) solution was prepared in Dulbecco’s Modified Eagle’s Medium (DMEM, glucose 4.5 g/L with phenol red) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate (Gibco), and penicillin (100 U/mL)-streptomycin (100 μg/mL) (Gibco). To examine NS LLPS dynamics, EGFP::SC35 MEFs (previously described in9) were treated with DMSO or 1 μM PP for 30 minutes and then treated with 0, 1, 2, or 10% 1,6-HD for 20 minutes. Cells were fixed in 2% paraformaldehyde, stained with bisBenzimide H 33258 (Hoechst), and then imaged. Image analysis was completed in Cell Profiler; briefly, the process was to image the cells in the 405 (Hoescht), 488 (GFP), and 555 (high intensity to image whole cells) and then use the 405 channel to determine the nuclei boundaries, 488 to determine EGFP::SC35, and 555 to determine the area of the whole cells. We differentiated nuclear and cytosolic areas by subtracting the 405 signals from the 555 signals. 488 signal was then quantified in the aforementioned nuclear area and cytosolic area and compared. 20+ cells were measured for each condition. The average Manders coefficient was determine with ImageJ116 by averaging the tM1 and tM2 values.

This same process was used to examine the effects of PP on GW182 and MED1 except in wildtype MEFs. The signal was identified by immunofluorescence (IF). Briefly, IF was performed by fixing cells with 4% paraformaldehyde in PBS, permeabilizing with 0.2% Triton X-100 in PBS, blocked with 2% bovine serum albumin in PBS, and then incubated with primary antibody (GW182 (ab156173, Abcam) or MED1 (ab60950, Abcam)) diluted per manufacturer’s recommendation overnight at 4°C. Cells were then treated with the appropriate 1:1000 secondary antibody overnight at 4°C, stained with Hoechst the following day, and then mounted with ProLong Gold Antifade (Invitrogen). Signal (either IF or endogenous GFP) sphericity was determined as previously described in9. 20+ cells were measured for each condition.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

For Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), cDNA was produced using the Superscript III (Thermo Fisher) kit and qPCR was completed using the SYBR Green system (Thermo Fisher) in a CFX384 Real-Time System (Bio-Rad). Endogenous β-actin levels were used as controls. The qPCR primer sequences were as follows:

β-actin forward: AAGGCCAACCGTGAAAAGAT

β-actin reverse: GTGGTACGACCAGAGGCATAC

Amot forward: CTGGAAGCAGATATGACCAAGT

Amot reverse: GGTGTTAGGAGAGTGGCTAATG

Atf4 forward: CCACTCCAGAGCATTCCTTTAG

Atf4 reverse: CTCCTTTACACATGGAGGGATTAG

Atg4c forward: GTGCGGAATGAGGCTTATCA

Atg4c reverse: CCAGACTTCTTCCCAAACTCTATC

Bmp4 forward: AACGTAGTCCCAAGCATCAC

Bmp4 reverse: CGTCACTGAAGTCCACGTATAG

Ern1 forward: TCCTAACAACCTGCCCAAAC

Ern1 reverse: TCTCCTCCACATCCTGAGATAC

Fdz1 forward: GAGATCCACCTTCCAGCTTTAT

Fzd1 reverse: CACTCCCTCTGAACAACTTAGG

Hyou1 forward: GAGGCGAAACCCATTTTAGA

Hyou1 reverse: GCTCTTCCTGTTCAGGTCCA

Manf forward: GACAGCCAGATCTGTGAACTAAAA

Manf reverse: TTTCACCCGGAGCTTCTTC

Rnf166 forward: GAAGACACACTCCCGCTTTA

Rnf166 reverse: CTGAGACCAACTCTCCTTGTG

Sirt2 forward: CATAGCCTCTAACCACCATAGC

Sirt2 reverse: GTAGCCTGTTGTCTGGGAATAA

Sqstm1 forward: AACAGATGGAGTCGGGAAAC

Sqstm1 reverse: AGACTGGAGTTCACCTGTAGA

Tgfb3 forward: CCACGAACCTAAGGGTTACTATG

Tgfb3 reverse: CTGGGTTCAGGGTGTTGTATAG

Ube2q2 forward: TTCCTAAGCACCTGGATGTTG

Ube2q2 reverse: CTCCTCCTCTTCCTCTTCTTCT

Xbp1 forward: GGGTCTGCTGAGTCC

Xbp1 reverse: CAGACTCAGAATCTGAAGAGG

Cul5 forward: GAACACAGGCACCCTCATATT

Cul5 reverse: AGTTACACTCTCGTCGTGTTTC

Psmg1 forward: CCAGTGGTTGGAGAAGGTTT

Psgm1 reverse: GGGTCTTGTAGTCTGTGATGTG

Atg14 forward: CATTCCCTGGATGGGCTAAA

Atg14 reverse: CCTCAGGAACAAGAAGGAAGAG

Yap1 forward: CCAATAGTTCCGATCCCTTTCT

Yap1 reverse: TGGTGTCTCCTGTATCCATTTC

Chromatin Immunoprecipitation (ChIP)

ChIP for SC35 was performed using anti-SC35 antibody (ab11826, Abcam) as previously described117. Briefly, mouse liver samples were submerged in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration). The tissue pieces were then treated with a TissueTearer and finally spun down and washed twice in PBS. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. The chromatin was enzymatically digested with MNase. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, Proteinase K and heated for reverse-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. An aliquot of chromatin (10 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of antibody. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinking was reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP-qPCR for MEFs were essentially performed the same way as previously described with anti-SC35 (ab11826, Abcam) and anti-XBP1s antibody (Biolegend 658802), except that the MEFs were directly fixed with 1% formaldehyde before subject to nuclei isolation and chromatin immunoprecipitation. The primers used for ChIP-qPCR are as follows:

Gene desert forward primer: GCAACAACAACAGCAACAATAAC

Gene desert reverse primer: CATGGCACCTAGAGTTGGATAA

Xbp1 promoter region forward primer: GGCCACGACCCTAGAAAG

Xbp1 promoter region reverse primer: GGCTGGCCAGATAAGAGTAG

Xbp1 gene body region forward primer: CTTTCTCCACTCTCTGCTTCC

Xbp1 gene body region reverse primer: ACACTAGCAAGAAGATCCATCAA

Manf promoter region forward primer: ACAGCAGCAGCCAATGA

Manf promoter region reverse primer: CAGAAACCTGAGCTTCCCAT

Manf gene body region forward primer: CAACCTGCCACTAGATTGAAGA

Manf gene body region reverse primer: AGGCATCCTTGTGTGTCTATTT

Hyou1 promoter region forward primer: GACTTCGCAATCCACGAGAG

Hyou1 promoter region reverse primer: GACTTCTGCCAGCATCGG

Hyou1 gene body region forward primer: TGGAAGAGAAAGGTGGCTAAAG

Hyou1 gene body region reverse primer: TCCCAAGTGCTGGGATTAAAG

HTS of FDA-approved drugs and quantitative imaging analysis

EGFP::SC35 MEFs were seeded in black 384 well plate with glass bottom (Cellvis). The FDA-approved compound library (100nL per drug) was stamped to 384-well tissue culture plates using CyBio Well vario (Analytik Jena). Compound solutions were then added to the cell plate at the final concentrations of 10 μM using a BRAVO liquid handler. After 18 hours of treatment, culture media was removed, and cells were fixed with 4% PFA followed by DAPI staining. Cells were imaged using a GE INCELL 2200 with 60x lens. Maximum intensity projection images were captured, and nuclear speckles sphericity was quantified with CellProfiler as previously described in9. Briefly, for speckle i the sphericity is defined as equation 1:

Sphericityi=2π*areai÷circumferencei (1)

So that a perfect circle will have a sphericity of 1, and a line will have a sphericity of 0. To calculate the average sphericity of a given image that has k total speckles, we calculated the area-weighted average as described in equation 2.

Averagesphericity/image=1kSphericityi×areai/1kareai (2)

For the secondary screening to determine if compounds affected nuclear speckles morphology in a dose-dependent manner, specific compounds were selected using a TTP Mosquito X1 followed by serial dilutions of compounds that were prepared using a Bravo automated liquid handling platform (Agilent). Cells were treated, imaged, and analyzed according to the same protocol described above. For the tertiary screen we used MEFs with a Perk promoter-driven dGFP as previously described118. Cells were treated with drugs of interest and either DMSO or Tu at the same time as described above and both GFP/cell and cell number were determined with CellProfiler as previously described3. For both dose-response and Perk promoter-driven dGFP experiments, eight biological replicates were performed per drug per dose.

Immunofluorescence

Immunofluorescence was performed as previously described119. Briefly, liver OCT sections or cells cultured in chamber slide were fixed in cold acetone for 10 mins at −20 °C. The sections were then air dried, rehydrated with PBS and permeabilized with PBS+ 0.1% Triton X-100. The sections were then blocked with 10% goat serum at room temperature for 1 hour. For mouse liver tissues, primary antibodies against SC35/SRRM2 (ab11826, Abcam) were conjugated to Alexa-488, respectively per manufacture’s protocol and added to the OCT section at 1:1000 dilution overnight at 4 °C. Next day, sections were washed five times with PBS and counterstained with DAPI before mounting (with ProlongGold Glass) and imaging using Leica SP8 lightening confocal microscope (Leica Microsystems). For cell culture experiment, after incubation with anti-SRRM2 primary antibody, Alex488-conjugated secondary antibodies were added and the rest was performed as essentially the same way.

mRNA-seq and transcriptome analysis

For all mRNA-seq or RT-qPCR, total mRNA was isolated and purified from MEFs using the PureLink RNA Mini Kit (Thermo Fisher). For mRNA-seq, samples were submitted to the UPMC Genome Center for quality control, mRNA library preparation (Truseq Stranded mRNA (poly-A pulldown), and sequencing (paired-end 101 bp reads and ~40 million reads per sample). The sequencing was performed on a NextSeq 2000 sequencer.

For the son overexpression or knockdown (OE/KD) sequencing data, the raw RNA-seq FASTQ files were analyzed by FastQC for quality control. Adaptors and low-quality reads were filtered by Trimmomatic120. Then the processed reads were aligned by HISAT2121 against mouse reference mm10. For gene-level intron/exon quantification, bedtools software122 was used to collect and count reads that aligned to any intron/exon of the given gene. If one read spans across multiple exons of the same gene, it will only be counted once. If one read spans intron/exon junction, it will only be counted as intron. The intron/exon count was normalized by gene length and total reads for FPKM normalization.

The analysis of drug-treated samples was done using Galaxy123: Trim Galore was used for quality control124 and Salmon125 was used to normalize the paired-end reads (TPM method). The online 3D RNA-Seq126 pipeline was used to determine upregulated and downregulated genes, in addition to generating the PCA plot.

For RNA-seq of RhoP23H/+ and wild-type mice, total RNA was isolated from retinae isolated from mice at 1, 3, and 6 months of age using TRIzol organic extraction. RNA-seq was performed by QuickBiology Inc. RNA integrity was checked by Agilent Bioanalyzer. Libraries for RNA-seq were prepared with KAPA Stranded mRNA-Seq poly(A) selected kit (KAPA Biosystems, Wilmington, MA) using 250 ng toal RNAs for each sample. Paired end sequencing was performed on Illumina HighSeq 4000 (Illumina Inc., San Diego, CA).

The reads were first mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0127 and the gene expression level was estimated using RSEM v1.2.15128. TMM (trimmed mean of M-values) was used to normalize the gene expression. Differentially expressed genes were identified using the edgeR program129. Genes showing altered expression with p < 0.05 and more than 1.5-fold changes were considered differentially expressed. Goseq was uesd to perform the GO enrichment analysis and Kobas was used to perform the pathway analysis.

mRNA splicing rates analysis

The mRNA processing rate was estimated by the simple kinetic model (equation 3) where pre-mRNA was converted to mature mRNA with the mRNA processing rate Kp and the mature mRNA is subject to decay with a constant decay rate Kd.

PremRNAKpmaturemRNAKd (3)

Under the basal condition (DMSO), we assume a steady state of mature mRNA expression whose level does not change over time; thus, we have:

dcmaturemRNAdt=Kp×Cpre-mRNA-CmaturemRNA×Kd=0
Kp=C(maturemRNA)×KdC(PremRNA) (4)

Under Son overexpression or knocking down condition (condition 2), if we assume the mature mRNA degradation rate does not change with Son OE/KD (K1d=K2d), then the ratio of splicing rates between basal condition 1 and Son OE/KD condition 2 is given by:

Kp1Kp2=C1(maturemRNA)×C2(PremRNA)C1(PremRNA)×C2(maturemRNA) (5)

Gene set enrichment analysis (GSEA)

GSEA was performed with software version 4.1.0. TPM quantification of transcriptome under different drugs or DMSO control was used as input for gene expression. Parameters used for the analysis: 1000 permutations, permutation type: gene set.

Intron retention detection

Intron retention events were detected by iREAD31. Intron retention events are selected with default settings T>=20, J>=1, FPKM>=2.

Gene ontology analysis

DAVID (Version 2021)130 (https://david.ncifcrf.gov) was used to perform Gene Ontology analyses. Briefly, gene names were first converted to DAVID-recognizable IDs using Gene Accession Conversion Tool. The updated gene list was then subject to GO analysis using all Homo Sapiens as background and with Functional Annotation Chart function. GO_BP_DIRECT, KEGG_PATHWAY or UP_KW_BIOLOGICAL_PROCESS was used as GO categories. Only GO terms with a p value less than 0.05 were included for further analysis.

Motif analysis

Motif analysis was performed with the SeqPos motif tool (version 0.590)131 embedded in Galaxy Cistrome using all motifs within the homo sapiens reference genome hg19 as background. LISA analysis was performed using webtool (http://lisa.cistrome.org/).

Statistical Analysis

Data was analyzed and presented with GraphPad Prism software. Plots show individual data points and bars at the mean and ± the standard error of the mean (SEM). Individual tests used were provided at the end of each figure legend.

Supplementary Material

Supplement 1

Table S1. FPKM normalization of RNA-seq of SON OE and KD cells in the absence or presence of Tunicamycin in MEFs.

media-1.xlsx (5.8MB, xlsx)
Supplement 2

Table S2. TPM normalization of RNA-seq of different chemical treatments in MEFs.

media-2.xlsx (3MB, xlsx)
1

Acknowledgements

We would like to thank Drs. Yvonne Eisele, Yuan Liu and Toren Finkel (University of Pittsburgh School of Medicine, Pittsburgh, PA, U.S.A.) for their technical assistance and/or comments on the manuscript. We thank Dr. Krzysztof Palczewski who generously shared the 1D4 anti-rhodopsin antibody. W.D. was supported by grant T32 HL082610 through the National Institutes of Health (NIH), the Diana Jacobs Kalman/AFAR Scholarship for Research in the Biology of Aging through the American Federation for Aging Research, and fellowship F31 AG080998. B.B.C. was supported by grant 1R35HL139860 through the NIH. Y.C was supported by the R01 EY030991. X.C. was supported by the National Institutes of Health grants R01AG074273 and R01AG078185. B. Zhu was supported by grants 1DP2GM140924 and 1R21AG071893 through the NIH, and a grant from Richard King Mellon foundation. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. This research project was supported in part by the Pittsburgh Liver Research Centre supported by NIH/NIDDK Digestive Disease Research Core Center grant P30DK120531, the Ophthalmology and Visual Sciences Research Center core grant P30 EY08098, the Eye and Ear Foundation of Pittsburgh and an unrestricted grant from Research to Prevent Blindness.

Footnotes

Declaration of interests

B.B.C. is Co-founder of Koutif Therapeutic Inc., Co-founder and VP of Drug Discovery for Generian Pharmaceuticals, and Co-founder and C.S.O. of Coloma Therapeutics Inc..

Data availability

All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE224275. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD050371. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1

Table S1. FPKM normalization of RNA-seq of SON OE and KD cells in the absence or presence of Tunicamycin in MEFs.

media-1.xlsx (5.8MB, xlsx)
Supplement 2

Table S2. TPM normalization of RNA-seq of different chemical treatments in MEFs.

media-2.xlsx (3MB, xlsx)
1

Data Availability Statement

All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE224275. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD050371. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.


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