Pyrvinium to reduce tauopathy

Abstract

Pyrvinium compounds, or a pharmaceutically acceptable salt thereof, used in methods for reducing tauopathy in a subject in need thereof. Pyrvinium compounds, or a pharmaceutically acceptable salt thereof, used in methods for treating a disease associated with accumulation of misfolded proteins (e.g., proteinopathies, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, retinitis pigmentosa such as retinitis pigmentosa with rhodopsin mutations, aging, neurodegenerative diseases, or metabolic disorders).

Description

PYRVINIUM TO REDUCE TAUOPATHYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 519,112 filed on August 11, 2023, which application is incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on , 20 , is named SL.xml and is bytes in size.GOVERNMENT SPONSORSHIP

[0003] This invention was made with government support under AG071893 and GM140924 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Proteinopathies are diseases associated with the accumulation of misfolded proteins, which often arise from a decline in proteostasis pathways, including the ubiquitin- proteasome system, the ER-Golgi protein secretory pathways, and lysosomal uality control. However, therapies targeting singular pathways have limited efficacy, indicating an incomplete understanding of disease mechanisms . Thus, a need exists in the art to treat these conditions and this disclosure satisfies this unmet need.SUMMARY

[0005] In the present invention, a pyrvinium compound (e.g., pyrvinium pamoate) is provided as a small-molecule nuclear speckle rejuvenator. In particular, the pyrvinium compound (e.g., pyrvinium pamoate) can enhance protein quality control while suppressing YAP1 signaling via increasing the surface tension of nuclear speckle condensates through direct binding to nuclear speckle scaffold protein SON.

[0006] In an aspect, provided herein is a method of reducing tauopathy, the method comprising, or consisting essentially of, or consisting of administering to a subject an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof.

[0007] In another aspect, provided herein is a method of treating a disease associated with accumulation of misfolded proteins (e.g., proteinopathies, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, retinitis pigmentosa such as retinitis pigmentosa with rhodopsin mutations, aging, neurodegenerative diseases, or metabolic disorders), the method comprising, or consisting essentially of, or consisting of, administering to a subject an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figs. 1A-1L. Genetic rejuvenation of nuclear speckle transcriptionally reprograms global proteostasis and YAP1 signaling in an opposing manner.

[0009] Figs 2A-2I. HTS identifies pyrvinium as an SON-dependent nuclear speckle rejuvenator.

[0010] Figs. 3A-3O. PP targets SON to reduce the interfacial tension of nuclear speckles.

[0011] Figs. 4A-4O PP reduces pathological Tau and Rhodopsin level by boosting autophagy and UPS activity.

[0012] Figs. 5A-5M. PP alleviates proteinopathies in preclinical models.

[0013] Figs. 6A-6J. PP rejuvenates nuclear speckles and alleviates tau burden in human iPSC-neurons expressing mutant Tau.

[0014] Fig. 7. Proposed mechanism of action of PP.

[0015] Figs. 8A-8D. PP reduces tau burden in neurons by enhancing autophagy.

[0016] Figs. 9A-9B. PP treatment does not induce cell toxicity and global translational suppression in neurons.

[0017] Figs. 1A-1L. Genetic rejuvenation of nuclear speckle transcriptionally reprograms global proteostasis and YAP1 signaling in an opposing manner. (1 A) PCA analysis of global transcriptional response to Tu in the presence of SON OE (via CRISPRa) or KD (viasiRNA). (IB) Relative expression (R.E.) of representative proteostasis genes (left) and YAP1 target genes (right) in response to Tu in the presence of SON OE / KD (n=4 for SON KD, and n=3 for SON OE). (1C) 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. (ID) GO analysis of those 461 and 901 genes showing enriched KEGG pathways. (IE) Enriched XBP Is binding motif ACGTCA on the promoters of 461 genes. (IF) Top enriched GO terms for top 500 most abundant proteins that are detected in hepatic XBP Is interactome at CT8, as previously reported io. (1G) Heatmap showing relative abundance of 45 proteins involved in mRNA splicing and processing within the XBP Is interactome at CTO and CT8. respectively (p value of 3.5E-5 calculated by paired t-test indicating reduced abundance at CTO). (1H) Enriched TEAD2 binding motif GGCGG on the promoters of 901 genes. Western blot (II) and quantification (1J) of nuclear and cytosolic level of YAP1 in control and SON OE cells in response to Tu (n=2). Scratch assay with representative images (IK) and quantification (IL) 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.).

[0018] Figs. 2A-2I. HTS identifies pyrvinium as an SON-dependent nuclear speckle rejuvenator. (2A) 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. (2B) Dose-dependent effect on nuclear speckles morphology by PP, with a representative image of nuclear speckle under I pM of PP (n=10~16). (2C) Quantification of total area-normalized perimeter of nuclear speckles in control and 1 pM PP condition per cell (n=26 for DMSO and n=35 for PP). (2D) GSEA analysis showing a similar transcriptome signature between PP-upregulated genes and 461 genes further amplified by SON OE during ER stress. (2E) GSEA analysis showing a similar transcriptome signature between PP-downregulated genes and 901 genes further blunted by SON OE during ER stress. (2F) 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. (2G) GO analysisof differentially expressed genes by PP. (2H) 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 24 hours. (21) Logz normalized fold change in response to PP treatment (0.3 pm) 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.

[0019] Figs. 3A-3O. PP targets SON to reduce the interfacial tension of nuclear speckles. CETSA of SON (3 A) and GFP::SRSF2 (3B) with 3pM PP. Both representative blot and quantification from independent replicates are shown. P value is calculated by mixed- effects analysis (SON: n=6 for DMSO and n=5 for PP; GFP::SRSF2: n=3 for both DMSO and PP). (3C) Computational prediction of IDR in mouse SON. (3D) Diagram illustrating how surface tension influences droplets coalescence kinetics. (3E, 3F) Representative images of droplet formation assay with different recombinant proteins (3E) and quantification (3F) of area-normalized perimeter changes in the time span of 20 minutes with 125mM NaCl (n=2~3). (3G-3I) Representative images of droplet formation with NE-supplemented SON IDR2 with increasing concentration of PP (3G) and quantification of sphericity (n=12 for with NE and n=42 for without NE) (3H) and area-normalized perimeter changes (n=2) (i) in the time span of 20 minutes. (3J-3M) 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 (3J). Representative images (3K) and quantification of sphericity (31) and spatial distribution of mCherry::SON IDR2 and GFP::SRSF2 (3M). (3N, 30) Mouse genomic DNA was further added to the solution. Representative images (3N) and quantification (30) of spatial distribution of mCherry::SON IDR2, GFP::SRSF2 and DNA. All data mean ± S.E.M.

[0020] Figs. 4A-4O. PP reduces pathological Tau and Rhodopsin level by boosting autophagy and UPS activity. (4A-4F) MEFs were treated with vehicle control or IpM PP for ~24 hours (22 hours for 4E and 4F) and then co-treated with or without puromycin (10 pg / mL for 30 minutes), MG132 (lOpM for 110 minutes) or Baf A (lOOnM for 22 hours) (n=3 for all samples). Western blot and quantification of puromycin-incorporated proteins (4A, 4B), poly- ubiquitinated protein (4C, 4D) and LC3II and LC3II / LC3I ratio (4E, 4F). (4G-4H) NIH3T3 RHOP23H cells were treated with 0.1 pM PP for 24 hours and Western blot (4G) and quantification (4H) of RHOP23H level (n=3). (41, 4J) NIH3T3 RHOP23H cells were co-treatedwith or without MG132 (lOpM for 120 minutes). Western blot (41) and 40 quantification (4J) of RHOP23H level (n=3). (4K) qPCR of proteostasis and YAP 1 target genes in P301S-expressing primary neurons treated with or without PP (O.lpM) for 12 hours. (4K-4M) Tau (P301S)- expressing primary neurons were treated with increasing concentration of PP for 24 hours, and w estern blot and quantification of different proteins (n=4 for HT7, PHF 1 and LC3, and n=6 for P62) (4K) or treated with 0. 1 pM PP for 12h hours in the presence or absence of BafA (50nM) in the last hour and western blot (4L) and quantification (n=4 for total Tau and P62 and n=8 for p-Tau) (4M) of different proteins. (4N) Tau P301S-expressing neurons were treated with DMSO or O. lpM PP for 12 hours and qPCR of selective proteostasis and YAP1 target genes (n=6~l 1). (40) MEFs were treated with DMSO, PP (IpM), XMU-MP-1 (IpM) or XMU-MP- 1+PP for 24h hours, and qPCR of protein quality control and YAPls 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.

[0021] Figs. 5A-5M. PP alleviates proteinopathies in preclinical models. (5A-5E) RNA-seq was performed in the retina of 1 and 3 months-old wild-type and Rho P2 H / +mice. GO of differentially expressed genes (DEG) (FDRO.l) (5 A, 5C) and GSEA comparing these DEG with that of PP (5B, 5D). Heatmap of selective genes (5E). (5F-5J) 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. (5F) 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 pm (bottom). Scale, 1 mm. (5G) and (5H) are bar plots of retinal thickness and volume, respectively, measured from the vis-OCT scanning data where **** indicates P<0.0001 by two-way ANOVA and Turkey multiple comparison. Data: Means ±SDs. N=4-5. (51) Representative retinal histology images of the retina explants cultured for 10 days with black bars showing the outer nuclear layer (ONL). Scale, 20 pm. (5 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). Data: Mean ± SD. N=3-4. ***. P<0.001 by two-way AV OVA comparing Rhop23H / + retinal explants treated with PP vs. vehicle control. (5K-5M) C155>UAS-hTaul. l3 and Cl 55 flies were fed with either standard diet or diet supplemented with 25pM PP. Quantification of distance travelled from larval crawling assay(n=l 0) (5K). climbing index score from adult fly climbing assay at 14 days and 21 days (n=5) (5L) of age. Western blot and quantification of p-Tau (AT8 antibody) and total Tau (DAKO antibody) level in C155>UAS-hTaul. 13 flies fed with control or PP diet (5M).

[0022] Figs. 6A-6J. PP rejuvenates nuclear speckles and alleviates tau burden in human iPSC-neurons expressing mutant Tau. (6A) GO analysis of up and downregulated genes in 4R-Tau P301S iPSC neurons reported in 73. (6B) GSEA comparing genes upregulated in 4R-Tau P301S iPSC neurons with those downregulated by PP. (6C) Motif analysis of promoters of genes upregulated in 4R-Tau P301 S iPSC neurons compared to 4R-Tau (FDR<0.05) revealed top enriched motif of TEAD2. (6D, 6E) Wild-type and V337M Tau- expressing iPSC-neurons were treated with DMSO or PP (lOnM) for 12 hours, and IF against nuclear speckle (Ab 11826 against SRRM2), p-Tau (Ser422) and chromatin (DAPI) were performed. Representative images (5D) 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 (6E). (6F, 6G) V337M Tau-expressing iPSC-neurons were treated with DMSO or increasing concentrations of PP for 12 hours. Representative western blot (6F) and quantification (6G) of total and p-Tau (Ser202 / Thr205) (n=3). (6H) V337M Tau-expressing iPSC-neurons were treated with DMSO or lOOnM PP for 12 hours, and autophagy flux was quantified by the autophagy reporter. (61) PP is a HORMINAM by inducing stress (via mitochondria) as well as amplifying the stress response (via SON). (6J) Diagram showing relationship between stress and stress response under different conditions. PP can elicit the most robust stress response relative to stress. Data: Mean ± SEM.

[0023] Fig. 7. Mechanism of action of pyrvinium pamoate (PP) Via a cell-free nuclear speckle reconstitution assay, we identified the mechanism of action of PP is by reducing the surface tension of nuclear speckle condensates, via binding to the intrinsically disordered region (IDR) of the nuclear speckle scaffold protein SON. Simply speaking, we identified 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 (a subunit of the spliceosome complex) content at the periphery, increased wetting of genomic DNA and subsequently causing a higher portion of spliceosomes stably engaging in active RNA processing and transcription elongation of proteostasis genes.

[0024] Figs. 8A-8D. PP reduces tau burden in neurons by enhancing autophagy. (8 A) Representative images of SRRM2 IF and quantification of nuclear speckle sphericity and area in control and V337M 3R-Tau iPSC-neurons treated with DMSO or lOnM PP. (8B, 8C) V337M Tau iPSC-neurons were treated with DMSO or increasing concentrations of PP for 12h. Representative western blot (8B) and quantification (8D) of total and p-Tau (Ser202 / Thr205) (n=3). (8D) V337M Tau iPSC-neurons were treated with DMSO or lOOnM PP for 12h, and autophagy flux was quantified by the autophagy reporter.

[0025] Figs. 9A-9B. PP treatment does not induce cell toxicity and global translational suppression in neurons. (9A) LDH activity in the media of iPSC-neurons treated with DMSO or 0.5 pM PP for 24h. Media from triton-x lysed neurons were included for the maximum amount of LDH release. (9B) Western blot showing levels of p-elF2a. total elF2a and actin in lysates of WT and V337M iPSC-neurons treated with increasing concentration of PP for 24h.

[0026] Figs. 10A-10C. PP feeding ameliorates tauopathy phenotypes in flies. (10A) Larva crawling and (10B) adult fly climbing assays in control and Tau flies fed with Ctrl or PP- supplemented diet. (10C) Western blot and quantification of p-Tau (AT8) and total Tau (DAKO) levels in brain lysates of C155>Tau flies fed with control or PP diet.

[0027] Figs. 11A-11B. The potential of PP for treating neurodegeneration in mammals. (HA) PK of PP in mice with concentration in plasma and whole brain tissues different times after IP injection. (1 IB) qPCR in hippocampus of mice IP-injected with PP (0.5mg / kg) at different times and qPCR of Son level in the hippocampus of mouse at different times.DETAILED DESCRIPTION

[0028] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherw ise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0029] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” - for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0030] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example. “A, B. and / or C” would mean “A, B, C. A and B, A and C, B and C. or the combination of A, B, and C.”

[0031] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0032] Dose(s): As used herein, the term "dose(sf ’ means a quantity of the compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof to be administered at one time. A dose may comprise a single unit dosage form, or alternatively may comprise more than a single unit dosage form (e.g., a single dose may comprise two tablets), or even less than a single unit dosage form (e.g.. a single dose may comprise half of a tablet). A dose described herein may be administered at various intervals. For example, a dose as described herein may be administered daily or weekly.

[0033] Daily dose: As used herein, the term ‘'daily dose” means a quantity of the compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof that is administered in a 24-hour period. Accordingly, a daily dose may be administered all at once (i.e., once daily dosing) or alternatively the daily dosing may be divided such that administration of the compound is twice daily, three times daily, or even four times daily,

[0034] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or "reduce", or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control sample or subject (or multiple control samples or subjects) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0035] In Vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0036] In Vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0037] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g.,mammals such as mice. rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.

[0038] Preventing: The term "prevent," "preventing," or "prevention," as used herein refers to an effect that mitigates an undesired effect, e.g., an undesirable drug-drug interaction or the formation of a drug-iron chelate. Prevention does not require the 1.00% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced by the compound or method.

[0039] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one-unit dose.

[0040] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. In one aspect, prevention is excluded from the terms “treat,” “treatment” or “treating.”

[0041] Unit dosage form(s): the term “unit dosage form(s)” includes tablets; caplets; capsules, such as soft elastic gelatin capsules; sachets; cachets; troches; lozenges; dispersions; powders; solutions; gels: liquid dosage forms suitable for oral or mucosa I administration to a patient, including suspensions (e.g.. aqueous or non-aqueous liquid suspensions), emulsions (e.g., oil-in-water emulsions, or a water-in-oil liquid emulsion), solutions, and elixirs; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for oral or parenteral administration to a patient. The unit dosage form does not necessarily have to be administered as a single dose nor does a single unit dosage form necessarily constitute an entire dose.

[0042] Pharmaceutically acceptable: The term "pharmaceutically acceptable", as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0043] Pharmaceutically acceptable salts: pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g, alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2. Mg2+, Zn2+), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0044] Pharmaceutically acceptable carriers: “pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol.sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0045] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukary otic cell.

[0046] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0047] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.

[0048] “Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0049] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry’ is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0050] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0051] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabicnumerals referring to referenced citations, the full bibliographic details of which are provided subsequent to the Examples section. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the present technology.

[0052] As described herein, a pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof can serve as a small-molecule nuclear speckle rejuvenator. In particular, the pyrvinium compound or a pharmaceutically acceptable salt thereof can reduce tauopathy.

[0053] Accordingly, provided herein are methods for reducing tauopathy (e.g., reducing Tau levels such as total Tau levels and / or hyperphosphorylated Tau levels), the method comprising, or consisting essentially of, or consisting of administering to a subject in need thereof an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof. The subject is any animal, mammal or human in need of such reduction.

[0054] Also provided are methods of treating a disease associated with accumulation of misfolded proteins, the method comprising, or consisting essentially of, or consisting of administering to a subject in need thereof an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof. The subject is any animal, mammal or human in need of such reduction.

[0055] For example, the disease described herein comprises proteinopathies,Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), retinitis pigmentosa (RP), retinitis pigmentosa (RP) with rhodopsin mutations, aging, neurodegenerative diseases, cancers, and metabolic disorders.

[0056] In another aspect, provided herein are methods of one or more or all of: modulating global proteostasis, enhancing protein quality control, suppressing YAP1 signaling, enhancing the propensity of nuclear speckle liquid-liquid phase separation (LLPS), improving Son gene expression, reducing the surface tension and / or increasing the surface areas of nuclear speckles, upregulating protein quality control gene expression, downregulating YAP1 transcription output, reducing tauopathy, promoting autophagy, promoting ubiquitin- proteasome system (UPS) activity7, and preventing retina degeneration, the method comprising, or consisting essentially of, or yet further consisting of administering to a subject an effectiveamount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof. The subject is any animal, mammal or human in need of such reduction.

[0057] In some embodiments, the pyrvinium compound is administered in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical carrier.

[0058] In some embodiments, the subject is a mammal, e.g., an animal model such as a murine, rat, simian or pet, e.g., a canine, feline, equine, or farm animal such as a bovine, equine, or porcine. In some embodiments, the subject is a human patient.

[0059] In some embodiments, the pyrvinium compound is pyrvinium pamoate.

[0060] For the above methods, an effective amount may be administered to a subject such as a human patient. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, intratumoral, epidural and intrathecal. In some embodiments, administration can be intravenously, intrathecally, intraperitoneally, intramuscularly, subcutaneously, or by other suitable means of administration. The quantity and frequency of administration of each agent will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. Administration can vary with the subject and purpose of the therapy, e.g., in one aspect as an animal model to test or treat additional or combination therapies, or as a personalized model to treat a patient. Alternatively, the treatment is for veterinarian use.Compositions

[0061] In another aspect, a composition is provided that comprises, or consists essentially of, or yet further consists of a pyrvinium compound described herein (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or one or more excipients, fillers or agents (collectively referred to hereafter as ‘"pharmaceutically acceptable carrier7’ unless otherwise indicated and / or specified). In a related aspect, a medicament for treating a diseased as described herein is provided that includes a pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptablesalt thereof and optionally a pharmaceutically acceptable carrier. The medicament of any embodiment herein may include an effective amount of the pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof for use in a method described herein (e.g., for treating a diseased described herein). In any aspect or embodiment disclosed herein, the effective amount may be determined in relation to a subject.

[0062] The pharmaceutical composition may be packaged in unit dosage form. The unit dosage form may be effective for use in a method described herein (e.g., for treating a diseased described herein). Generally, a unit dosage including the pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof will vary' depending on patient considerations. Such considerations include, for example, age. protocol, condition, sex, extent of disease, contraindications, concomitant therapies and the like. An exemplary’ unit dosage based on these considerations may also be adjusted or modified by a physician skilled in the art. Suitable unit dosage forms, include, but are not limited to parenteral solutions, oral solutions, powders, tablets, pills, gelcaps, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained-release formulations, mucoadherent films, topical varnishes, lipid complexes, liquids, etc.

[0063] The pharmaceutical compositions and medicaments may be prepared by mixing the pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof and / or compositions of the present technology7with pharmaceutically acceptable carriers, excipients, binders, diluents or the like. Such compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The instant compositions can be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular, injections.

[0064] Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant present technology. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

[0065] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Generally, pyrvinium pamoate can be administered in dosages of between about O.Olmg / kg to 10 mg / kg; 0.04mg / kg to 5mg / kg; or 0. Img / kg to Img / kg.

[0066] The pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof can also be administered to a patient along with other conventional therapeutic agents that may be useful in the treatment of a disease as described herein. The administration may include oral administration, parenteral administration, or nasal administration. In any of these embodiments, the administration may include intratumoral injections, subcutaneous injections, intravenous injections, intraperitoneal injections, or intramuscular injections.

[0067] Also described herein are kits comprising, or alternatively consisting essentially of, or yet further consisting of one or more of: a pyrvinium compound (e.g., pyrvinium pamoate) or a pharmaceutically acceptable salt thereof, one or more optional naturally- occurring or non-naturally-occurring carrier(s), and optional instructions for use. In a further aspect, the instruction for use provide directions to conduct any of the methods disclosed herein.

[0068] The kit components, (e.g.. reagents) can be packaged in a suitable container. The kit can also comprise, or alternatively consist essentially of, or yet further consist of, e.g., a carrier such as a buffering agent, a preservative or a protein-stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

[0069] As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.EXAMPLES

[0070] Example 1

[0071] 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.

[0072] 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 cycle 3-13. By studying this 12h oscillator, we uncovered an unexpected role of nuclear speckles in global proteostasis control 9. Nuclear speckles are membrane-less organelles important for mRNA processing and gene regulation 14-18, and their liquid-liquid phase separation (LLPS) dynamics dictate the global transcriptional capacity of proteostasis genes 9. 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 aggregation 9. 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 aggregates 19. Since SON expression decreases with age in various tissues in mice, in human lung, and in brain tissues of human subjects with Alzheimer's disease (Extended Data Fig la-g) 9,20, we hypothesize that genetically or pharmacologically boosting SON expression and / or function (a term we herein coin as "‘nuclear speckles rejuvenation") could augment the entire proteostasis network and thus potentially delay or even reverse the progression of both aging-related and inherited proteinopathies.

[0073] 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 LLPS enhancement, unfolded protein response (UPR) transcription factors (TF)-mediated proteostasis gene activation and YAPl-mediated transcriptional repression. 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 domain (IRD) of SON. In preclinical models, PP exhibited strong efficacy in protecting against both tauopathy and retinal degeneration at nanomolar concentration. We further demonstrated that the previously known anti-YAP / WNT activity' of PP 21-23 could be explained by the expanded proteostasis framework and is also required for its ability to ameliorate proteinopathies. Lastly, we showed that both the reduction of proteostasis gene expression and increase of YAP1 transcriptional activity is associated with retinal degeneration in mice and tauopathy in humans.

[0074] Results

[0075] Genetic rejuvenation of nuclear speckle transcriptionally reprograms global proteostasis and YAP1 activity in an opposing manner.

[0076] To determine the extent by which SON transcriptionally reprograms gene expression under both basal and proteotoxic stress conditions, bulk mRNA-Seq was performed on immortalized mouse embryonic fibroblasts (MEFs) with either SON knockdown (KD) by siRNA or overexpression (OE) via CRISPRa24, in the absence or presence of the ER stress inducer tunicamycin (Tu) as previously described. 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. la). These include 461 genes that are normally induced, and 901 genes repressed by Tu under normal SON expression condition (Fig. Ib-lc). For both groups of genes, it was further observed a strong correlation between the relative fold induction or repression for each gene under SON OE and KD conditions, further demonstrating the robustness of bidirectional control on proteostasis gene expression by SON.

[0077] 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, Dnajbl l and Manf), ER / Golgi quality control (such as Sec23b, Hyoul and Hspa5), tRNA aminoacylation (such as Gars, lars and Eprs), ER-associated protein degradation (ERAD) (such as Edeml, Syvnl, Selll and Ube2g2), and autophagy' (such as Sqstml and Atgl3). 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) 25 and motif analysis to infer the transcriptional regulators that may recruit nuclear speckles to chromatin. Both analyses revealed basic leucine zipper (bZIP) transcription factors (TFs), including ATF6, XBP1, ATF4 and CREB1 as the top candidates (Fig. le). ChlP-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 XBPls recruitment to the promoter regions of the same genes. To corroborate the in vitro findings, we further examined a recently published murine in vivo hepatic XBPls interactome dataset 10, and found proteins involved in mRNA splicing and processing are very strongly enriched in the XBPls interactome at CT8. a time when hepatic SON expression peaks 9, with PRPF8, SNRNP200 and DHX9 being the top three most abundant proteins detected in the entire XBPls interactome at CT8 (Fig. If, 1g). By contrast, at CTO when SON expression is the lowest, the amount of splicing proteins that XBPls interacts with is markedly reduced (Fig. 1g). The observed decreased recruitment of splicing proteins to XBPls at CTO is not due to reduced XBPls level itself, as the hepatic XBPls expression at CTO is in fact higher compared to CT8 3,26. 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 XBPls.

[0078] Compared to induced genes, much less is known about the gene programs that are repressed under ER stress. GO analysis indicate that those 901 repressed genes are strongly enriched in Hippo-YAPl 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, Tubala, Fzd2, Tgfb3 and Yapl itself and its transcriptional partner Tead2 27-29 (Fig. I b-l d). YAP1 and TEAD2 were further predicted to be transcriptional regulators of these 901 genes via both LISA and motif analysis (Fig. Ih) and nuclear YAP1level was significantly reduced in response to either Tu or SON OE and further decreased upon the combination of the two (Fig. li, Ij). 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. Ik, 11). To rule out the possibility that the global repression of YAP 1 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) treatments 30, and observed a strong downregulation of genes involved in YAP 1 signaling under both treatments that progressed with time. Together, the data indicates that the downregulation of YAP 1 transcriptional output is an integral component of the global transcriptional response to proteotoxic stress, and it is also under nuclear speckles control.

[0079] Given the established roles of nuclear speckles in mRNA processing 31-33, 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 (consistent with total mRNA shown in Fig. la), it exerted profound effects on the pre-mRNA level, 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 algorithm 34, 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 YAP 1 -related processes of cell migration, axon guidance, cell adhesion and EMT. The significant enrichment in mRNA processing genes themselves under SON control is consistent with known potent autoregulation of splicing factors 35,36. 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. Finally, we observed that SON can also activate an anti-viral response gene signature under basal DMSO condition, 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 in vitro.

[0080] An expanded proteostasis framework includes nuclear speckle and YAP1.

[0081] To further confirm that the opposing dynamics of proteostasis gene expression and YAP1 transcriptional output also occur in vivo, we examined their temporal gene expression rhythms in mouse liver. In addition to proteostasis genes, which exhibit prevalent ~12h ultradian rhythms of expression as previously reported 3,6,9, we further identified fift - three hepatic ~12h-cy cling genes involved in Hippo-YAPl signaling, including Yap 1, Bmp4, Axin2, and Fzd6 / 7 / 8. More importantly, while proteostasis genes (including XBP Is itself) peak almost exclusively at CT2 and CT14, many Hippo-YAPl controlled genes, including nuclear YAP1 level itself, exhibit opposite phases peaking around CT6 and CT18. The anti-phase relationship between proteostasis and YAP 1 target genes under physiological condition in vivo is in line with their opposing transcriptional responses to acute ER stress in vitro.

[0082] The results thus far demonstrated a tripartite network where nuclear speckle rejuvenation by SON boosts proteostasis and suppresses YAP 1. Two possible topologies of the network exist. In model one, nuclear speckles can signal both proteostasis and YAP 1 signaling directly, while in model 2, nuclear speckles repress YAP1 downstream of increased proteostasis gene program. 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 XBPls small molecule activator IXA4 37-39. 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. 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 XBPls for the former, and triggering YAP1 nuclear exclusion for the latter . We speculate the opposing changes in proteostasis and YAP1 signaling may reflect an energetic trade-off between proteostasis and the control of cell dynamics, which could be essential for maintaining cellular functions in the presence of a fluctuating environment.

[0083] High-throughput screen (HTS) identified pyrvinium pamoate (PP) as a smallmolecule rejuvenator of the nuclear speckle.

[0084] Having established the proof-of-principle of nuclear rejuvenation via SON OE, we next explore the feasibility of rejuvenating nuclear speckles pharmacologically, for tworeasons. 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 speckles 19, respectively, putative nuclear speckle rejuvenators are expected to reduce the sphericity (and increase the diffuseness or irregularity) of speckles.

[0085] This 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 speckles 9) to identity’ compounds that could alter nuclear speckles sphericity (Fig. 2a). As a quality control, our primary7screen successfully identified four histone deacetylase inhibitors that produced much more spherical nuclear speckles approaching and / or exceeding r=0.9, in alignment with a previous study 40. 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 sphericity7and amplify7Perk-promoter driven dGFP expression (Perk is a UPR target and exhibits 12h rhythms of expression 3) in a dose-dependent manner (Fig. 2b). For PP, the reduced sphericity (more irregularity) is also further associated with increased perimeter of nuclear speckles (Fig. 2c), suggestive of a large surface area of nuclear speckles in the 3D space of the nucleus.

[0086] 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 with those of SON OE and KD under both DMSO and Tu conditions. Gene set enrichment analysis (GSEA) 41 using either those 461 Tu-induced or 901 Tu-repressed genes (depicted in Fig. 1c) 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). These included both upregulated genes implicated in protein quality7control and downregulated genes involved in the regulation of cell dynamics (Fig. 2f, g). LISA analysis on differentially expressed genes by PP revealed bZIP TFs ATF4 and YAP1 among toptranscriptional regulators of upregulated and downregulated genes, respectively. PP increased the expression of UPR and integrated stress response (ISR) TFs - XBPl s and ATF4 - at both the mRNA and protein level and induced nuclear exclusion of YAP1, while not affecting SON level.

[0087] The analysis so far suggests that 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). Secondly, similar to SON OE, PP also induced expression of genes involved in anti-viral response, 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 condition in the absence of ER stress. Lastly, like SON, PP also improves the splicing fidelity of splicing genes themselves again reflecting autoregulation of splicing factors. Taken together, these results indicate that PP is a bona fide nuclear speckle rejuvenator that induces a transcriptional signature in MEFs highly similar to that of SON OE cells under both basal and proteotoxic stress conditions.

[0088] PP reduces the surface tension of nuclear speckle condensates via targeting SON IDR.

[0089] To confirm that PP rejuvenates nuclear speckles in an SON-dependent manner, we knocked down Son via siRNA in MEFs. Son knockdown leads to smaller and more spherical speckles, consistent with our previous study 19 (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 capacity 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 stability 42,43. Using a SON-specific antibody, 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 speckleprotein, or the ISR / UPR TF ATF4, whose expression is nonetheless significantly increased by PP (Fig. 3b).

[0090] SON is the central scaffold protein of nuclear speckles 44-46, 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 state 9. 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 formation 47,48. In MEFs, PP generated a more diffuse and irregular (less spherical) nuclear speckles with larger surface area (Figs. 2b, 2c), suggesting that PP could influence nuclear speckle LLPS dynamics, likely via reducing the surface tension of speckle condensates (surface tension is the tendency of liquid droplets to minimize the total surface area, therefore an increased surface area is suggestive of reduced surface tension 49). 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 assay 50. Using different computational algorithms to search for intrinsically disordered domain (IDR) 51-53, 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). 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. Purified recombinant proteins were added to buffers containing 10% crowding reagents PEG-8000 as previously described 50. Confocal fluorescence microscopy of the different protein solutions revealed mCherry positive, micronsized spherical droplets freely moving in solution and wetting the surface of the glass coverslip. All proteins droplets were highly spherical, exhibited fusion / coalescence behaviors, and scaled in size and number positively with increasing concentration of proteins and negatively with increasing salt concentration, all properties expected for liquid-like droplets 50.

[0091] 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 ripening 54 (Fig. 3d), which was seen for all protein droplets after 20 minutes of time lapse imaging (Fig. 3e, 31). Addition of PP to SON IDR2, but not SON IDR1 and SRSF2protein 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. 3d-3f). 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.

[0092] To better recapitulate the complex compositions of nuclear speckles, we further supplemented recombinant mCherry-SON IDR2 with nuclear extract (NE) from Hela cells that include all active components of transcription and splicing factors 50. Mass spectrometry confirmed that Hela NE-supplemented mCherry-SON IDR2 condensates preferentially compartmentalized splicing factors (including SRSF2), but not other nuclear proteins like proteasome subunits, DNA repair factors or general transcription factors. These condensates further exhibited less spherical morphology, had increased number and total size, features expected from nuclear speckle-like condensates with inhomogeneous viscoelastic properties 55. Importantly, addition of PP reduced both the sphericity and surface tension of Hela NE- supplemented SON IDR2 condensates in a dose-dependent manner (Figs. 3g-3i), consistent with the 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.

[0093] To investigate whether PP may affect the relative spatial distribution of SON and SRSF2 within nuclear speckles, we further supplemented recombinant mCherry-SON 1DR2 with NE from GFP::SRSF2-expressing MEFs (Fig. 3j). The resulting nuclear specklelike 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. 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 (Figs. 3k-3m). To determine whether PP may also influence the wetting of genomic DNA by nuclear speckles 56, we further added mouse genomic DNA into the droplet solution. In accordance with observations in intact cells, nuclear speckle condensates largelydon’t mix with, but can wet the DNA. Interestingly, the addition of PP more than doubled the wetting of genomic DNA by the reconstituted nuclear speckles (Figs. 3n, 3o).

[0094] To validate that PP can alter the nuclear speckle LLPS properties in vitro in the context of 339 intact cells, we further performed 1,6 hexanediol sensitivity assay 57 using the same EGFP::SRSF2 MEFs 9. 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 condensates 57. As demonstrated in PP desensitized SRSF2 to increasing concentrations of 1 ,6 hexanediol. This effect was not observed on two other biomolecular condensates, the nuclear MED1 50 and cytosolic GW182 present in P- bodies 58. 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.

[0095] PP reduces both pathological Tau and rhodopsin levels by boosting ALP and UPS, at the 354 expense of YAP1 signaling.

[0096] 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 ALP and UPS in MEFs. Using puromycin incorporation assay 59, we found that in spite of increased expression of many mRNAs involved in ribosome biogenesis and translation (Fig. 2g), PP elicited a repression of global protein synthesis (Figs. 4a, 4b), an observation consist with the activation of the ISR 60. 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. PP led to a significant reduction of poly- ubiquitinated protein levels (Figs. 4c.4 d), and co-treatment with MG132 caused a one-fold increase of their level, while under vehicle condition, only a very small (statistically not significant) increase was observed (Figs. 4c, 4d). These data collectively indicated that PP increases UPS-mediated degradation of poly-ubiquitinated protein. To measure autophagicflux, we blocked autophagy at the late stage autophagosome-lysosome fusion step using Bafilomycin A 1 (BafA) 61 , and quantified the level of LC3I and LC3II with or without PP. 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 (Figs. 4e, 4f). These results indicate that PP also augments autophagic flux. In sum, these results revealed that PP represses global protein synthesis while promoting both ALP and UPS.

[0097] 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) mutations 62. To determine whether PP can protect against proteinopathies via boosting UPS and / or 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 rods and secondary loss of cones. Proline to histidine at codon 23 (P23H) is the most common mutation in RHO protein, resulting in autosomal dominant RP in humans 63. The heterozygous RhoP23H / + knock-in mouse develops progressive retinal degeneration that resembles the RP phenotype in patients 64,65. Recently, several studies suggested that boosting ERAD can protect against mouse models of RP by increased elimination of the mutant RHOP23H protein 64,66-68. To determine if PP can also reduce RHOP23H level, we used a NIH3T3 cell line ectopically expressing RHOP23H protein 69. Treating this cell line with 0. 1 pM of PP for 24 hours led to a reduction of both monomer and dimer forms of RHOP23H protein in an SON-dependent manner (Figs. 4g, 4h). Blocking the UPS, and to a lesser degree autophagy7, abolished the effect of PP on reducing RHOP23H protein level (Figs. 4i, 4j), suggesting that increased ERAD / UPS is likely mainly responsible for the increased elimination of RHOP23H by PP.

[0098] Both UPS and ALP are involved in the degradation of tau protein in tauopathies 70-73. 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) 74. 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 lOOnM PP (Fig. 4k). It is noteworthy that these reductions in Tau level occurred without anyobservable signs of cellular toxicity. PP also promoted autophagic flux in neurons, as demonstrated by an increase of LC3 TI / I ratio, decreased level of p62 / SQSTMl and increased autolysosome formation assayed with a duo-fluorescent autophagy7reporter (Fig. 4k) 75. Blocking autophagic flux with BafA dampened the effects of PP on reducing cellular Tau level (Figs. 41, 4m). As observed in fibroblasts, PP also promoted UPS activity in neurons; however, inhibiting proteasome activity with MG132 has minimal effects on PP’s ability7to reduce Tau level. Consistent with PP’s ability to boost UPS and ALP, PP increased the expression of genes involved in both pathyvays 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 neurons.

[0099] 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 yvhether 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 TRULI 76.77,78. 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, XMU-MP-1 negated PP’s effect on reducing RHOP23H level in NIH3T3 cells. Similarly, TRULI also dampens PP’s effect on reducing Tau level in primary neurons. 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 YAP 1 activity.

[0100] PP protects against mouse retina degeneration ex vivo and alleviates tauopathy in Drosophila.

[0101] Next, yve 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 (Figs. 5a-5e). In the retina of one month-old RhoP23H / + mice, yve observed a significant doyvnregulation of protein transport and autophagy7gene expression that showed large convergence with those upregulated by PP (Figs. 5a, 5b).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) 79. By three months, the downregulation of proteostasis gene expression persists in RhoP23H / + mice retina, concomitant with a significant increase of YAP 1 -mediated cell dynamics gene expression that also overlaps with PP-downregulated genes (Figs. 5c-5e). To directly tested 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) imaging 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 (Figs. 5f-5j ), 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 profile of PP as a promising therapeutic candidate.

[0102] 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 span 80. and SON 1DR2 is also conserved in flies. Thus, we next tested the efficacy of PP in ameliorating tauopathy in flies that express 2N4R isoform of human Tau (MAPT) pan-neuronally [elavcl55-Gal4: UAS-hTaul.13 (C155>UAS-hTaul. l3)] as well as in control elav cl55-Gal4 (C155) flies 81. Both C155>UAS-hTaul. l3 and C 155 flies were fed with either standard diet or diet supplemented with 25 pM PP, which did not affect the normal development and growth of flies despite its effect in attenuating WNT and YAP1 signaling 82. We quantified disease progression at different stages of fly development with both larv al crawling and adult fly climbing assay at 14 and 21 days of age. PP feeding preserved motor function in C155>UAS-hTaul. l3 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 (Figs. 5k, 51). Notably, PP also enhanced the motor function of adult wild-type control (Cl 55) flies at 21 days of age (Fig. 51). 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 flies 83. Consistent with the overall phenotypes, PP significantly reduced the level of p-Tau and to a lesser extent total Tau in the brains of C155>UAS-hTaul. l3 flies (Fig. 5m). Since p-Tau areprone 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.

[0103] PP has the potential for treating tauopathy in humans.

[0104] 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 subjects 84, and found that genes repressed by PP have increased expression in all four brain regions of human subjects with AD (such as YAP 1, TEAD1 and AMOT). By contrast, genes that were upregulated by PP displayed significantly decreased expression in temporal cortex (such as genes involved in ERAD: EDEMI, SEL1L, autophagy: ATG13, protein folding: 14Y0U1, and tRNA aminoacylation: GARS. IARS). 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' 85. 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. 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. This early to late AD progression is concomitant yvith strong increase of tauopathy, but not amyloid burden in these individuals. Consistent with in vivo data, we also observed a significant decrease of proteostasis gene expression as well as an increase ofYAPl- 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 (Figs. 6a-6c) 73. These upregulated YAP1-TEAD2 target genes also overlap with those repressed by PP (Fig. 6b).

[0105] 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 cells 86 and treated both cell lines with nanomolar range of PP for 12 hours. Immunofluorescence against nuclear speckles markerSRRM2 revealed that compared to controls. V337M iPSC-neurons exhibited aberrant nuclear speckle morphology characterized by smaller size and more spherical shape, and PP treatment fully restored nuclear speckle morphology' to normal size and diffuseness (Figs. 6d, 6e). Consequently, PP markedly reduced the level of p-V337M Tau via boosting the autophagic flux (Figs. 6d-6h). 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.

[0106] PP is a HORMINAM.

[0107] Taken together, our results indicate that the effects of PP are two-pronged. First, PP can induce modest stress to cells (Fig. 6i), likely due to its reported ability to trigger mitochondrial stress by increasing ROS levels and impeding mitochondrial functions 87-89. Mitochondria stress can activate the ISR and ATF4 activation 90.91, which likely explains the strong induction of ATF4 protein level by PP. Secondly, our new data showed that by directly binding to SON and reducing the surface / interfacial tension of nuclear speckles and promoting their wetting of DNA, PP is a bona fide nuclear speckle rejuvenator capable of amplifying the stress response at the transcriptional level, entailing both the upregulation of global protein quality control gene expression and downregulation of YAP 1 -mediated genes underlying cellular dynamics (Fig. 6i). In light of the dual effects of PP, we tentatively name it as a Hormesis Initiator and Amplifier (HORMINAM), a new class of compounds capable of both initiating and amplifying the hormetic response. A key characteristic of HORMINAM is its capacity to trigger a significantly stronger stress response relative to the actual stress it exerts, similar to the effect of SON OE in the presence of Tu (Fig. 6j). Our reclassification of PP as a HORMINAM thus reconciles the two previously known effects of PP: inducing mitochondrial stress and inhibiting YAP / WNT signaling 21,23. Intriguingly, a recent study using unbiased whole genome CRISPR screen to identify pyrvinium-genetic interactions revealed that knocking out spliceosome genes sensitized cells to pyrvinium-induced cell death 88. Although the underlying mechanism for this observation w as not addressed 88, our study herein provides a scientifically sound rationale for this observation: knocking out spliceosome genes abolishes the stress response-amplifying effect of pyrvinium without affecting its stress-inducing ability. The absence of a robust stress response following exposure to stress is anticipated to rendercells more susceptible to cell death. It is important to recognize that while pyrvinium acts as both a nuclear rejuvenator and HORMINAM, these concepts are independent of each other. It is conceivable that novel compounds could function solely as a HORMINAM without affecting nuclear speckle LLPS, or vice versa.

[0108] Discussion

[0109] Several recent studies indicate that both the decline of nuclear speckle functions and dysregulated mRNA splicing are associated with proteinopathies in humans, including both tauopathy and RP. For example, two studies showed that elevated Tau aggregates have the capability to relocate to the nucleus, thereby directly modifying the characteristics of nuclear speckles 92,93. In agreement with this study, a recently published Tau interactome in human iPSC neurons 94 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 is associated with neurofibrillary tangle burden in human AD subjects 95. 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 degeneration 96. These studies thus provide the rationale for nuclear speckle rejuvenation as a strategy for counteracting various proteinpathies.

[0110] Exploring the therapeutic potential of targeting biomolecule condensates represents an exciting avenue for research and drug development 97,98. Our study serves as a proof-of-principle showing 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.

[0111] Through a high-throughput drug screen, we identified PP as a small nuclear speckle rejuvenator by directly interacting with SON and modulating nuclear speckle LLPSdynamics. Interestingly, pyrvinium is enriched with cationic amines and aromatic motifs, chemical features that were predicted to partition into various nuclear condensates 99. PP was originally developed as an anthelmintic drug effective for treating pinworm infections 100. Moreover, it has gained strong recent interest as an anti-cancer reagent due to its ability to inhibit WNT signaling 100. 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. The possibility for the latter is made even more tantalizing due to its ability to cross blood brain barriers in mice.

[0112] While we don’t yet know the full detailed mechanisms by which PP modulates nuclear speckles dynamics and boost 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 chromatin 49. Thus, since spliceosomes reside at the interfacial boundary between nuclear speckles and nucleoplasm / chromatin47, 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 condensates 101,102. 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.

[0113] 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 YAP 1 -regulated genesinvolved 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 impeded cell migration. Additionally, the expressions of these genes follow anti-phasic ~12h ultradian rhythms in mouse liver under normal physiological conditions. These findings demonstrate that YAP 1 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 and ER stress.

[0114] 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 XBPls, leading to augmented transcription of proteostasis genes. Since the number of proteostasis genes under the control of SON surpasses those directly regulated by XBPls, 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 condensates 49. 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 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-localization 103, suggesting a low level of wetting of these two condensates under normal physiological conditions. Pending further investigation, we speculate herein that nuclear speckle rejuvenation may further reduce the wetting of these two condensates, resulting in the alteration of YAP condensate composition, and ultimately its nuclear exclusion.

[0115] 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 YAP 1 ortholog Y orkie, in the cytoplasm to restrict Y orkie transcription output 104. 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 autophagy 105. The latter is further consistent with our findings showing that restoring Y AP 1 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 activity7. Thus, a delicate balance between protein quality control and YAP1 activity7appears 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 studies 84,106. 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.

[0116] In conclusion, this 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, the findings underscore the significance of harnessing the 12-hour oscillator to unveil hidden principles of proteostasis control.

[0117] Materials and methods

[0118] Mice

[0119] 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).

[0120] Larva crawling assay

[0121] PP solubilized in DMSO were diluted directly into the fly medium at the final concentration of 25 pM 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.

[0122] Adult fly climbing assay

[0123] Male adult Drosophila melanogaster flies at 14 and 21 days of age fed with a normal diet or diet supplemented with 25 pM 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 enhancevisibility 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 w as taken to ensure the camera w as 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.

[0124] Fibroblast cell culture and drug treatment

[0125] 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 pg / 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 in 9. For Tu treatment, 100 ng / mL Tu (in DMSO) for six hours was used unless otherwise noted. NB (14Y-50904), PB (HY-12047), PH (HY-B0883), PP (HY-A0293), MG-132 (HY-13259), and XMU-MP-1 (HY-100526) were purchased through MedChemExpress and BafA (1334) was purchased from Tocris. All drugs were handled per manufacturer instruction.

[0126] Primary' neuron cell culture and P301S-Tau virus infection

[0127] The cerebral cortices of 3-4 neonate mice (P0) were dissected on ice, the meninges w ere removed, and placed in the cold dissection medium (DM), consisting of 6 mM MgC12 (SigmaM1028-100 ml), 0.25 mM CaC12 (Sigma C7902), 10 mM HEPEs (100X), 0.9% Glucose, 20 yM D-AP5 (Cayman, NC1368401). and 5 pM 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 w ater bath for 20 min. The suspension w as shaken every 5 min. lOmL 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 freshsolution was added in. Then the tissues were triturated until there were no visible chunks, and the solution was filtered through the 70 gm 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.

[0128] Human iPSC-derived neurons culture

[0129] Human iPSC-derived neurons were pre-differentiated and differentiated as described 86. 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, lx MEM non-essential amino acids, 1 x 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 pg / ml of mouse laminin (Thermo Fisher Scientific, cat. no. 23017-015), 10 nM ROCK inhibitor and 2 pg / 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 (Coming, 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, l x MEM non-essential amino acids. 0.5x GlutaMAX Supplement (Gibco / Thermo Fisher Scientific, cat. no. 35050-061), 0.5x N2 Supplement, 0.5x B27 Supplement (Gibco / Thermo Fisher Scientific, cat. no. 17504-044), 10 ng / ml of NT-3, 10 ng / ml of BDNF and 1 pg / ml of mouse laminin. Neuronal medium was halfreplaced every week.

[0130] Efficacy test of PP in Retina explant culture

[0131] Wild type and RhoP23H / + mice were euthanized at Pl 5 and retina explants were isolated and cultured as previously describedl07,108. 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 oC for 30 min. The digestion was stopped bytransferring the eyes to Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal calf serum (FCS; Gibco) and penicillin &streptomycin antibiotics (lx, GenClone) at 4 oC 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 oC with 5% CO2. The medium was replaced with fresh medium containing 0.5 pM PP after 24 h. which was replaced again even’ 2 days until 70 days in culture (DIV). A visible light optical coherence tomography (vis-OCT) prototype 109 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) stainingl 10. 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.

[0132] Autophagy reporter assay

[0133] 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.

[0134] Scratch assay

[0135] Cells were grown until 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 (Ohr) and then after 23hr. The Cell Profiler 111 “Wound Healing’" pipeline (https: / / cellprofiler.org / examples) was used to measure the “Percentage of Gap Filled”.

[0136] Immunoblot

[0137] 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 pg 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-a-Tubulin (Cell Signaling Technology (C.S.T.) #2144), anti-Lamin A / C (C.S.T. #4777), anti-SON (Abeam #121033 and LSBio LS-C803664), anti-YAPl (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-[3-actin (C.S.T. #4970), anti-puromycin (BioLegend 381502), antiubiquitin (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), and anti-XBPls (BioLegend 658802). 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 ImageJ 112.

[0138] Cellular thermal shift assay (CETSA)

[0139] EGFP::SC35 MEFs with EGFP knocked into the N-terminal of mouse Srsf2 locus (previously described in 9) were treated with either DMSO or 3pM PP for 50 minutes at 37°C. Cells were then trypsinized and resuspended in PBS with either DMSO or 3pM PP and 100 pL 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 nitrogenand thawed for three cycles to lyse the cells, and then spun at 20,000 x 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.

[0140] Protein purification and in vitro droplet formation assay

[0141] 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 grow th continued for 3h at 37°C. The cells w ere 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 lOmM 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 with rocking, then allowed to flow through the column. The packed agarose was washed with 15mL of Buffer A with l mM imidazole. Protein was eluted with 5mL of Buffer A containing 15mM imidazole, lOmL Buffer A containing lOOmM imidazole, then lOmL Buffer A containing 200mM imidazole. All elutions w ere collected in ImL fractions. Aliquots of the collected fractions were run on an SDS-PAGE gel and stained with Imperial Protein Stain to verify amount and purity of the protein. Fractions containing protein were combined and dialyzed against Dialysis Buffer (50mM Tris-HCl, 500mM NaCl, 10% glycerol, ImM 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. ImM DTT, 10% PEG) containing indicatedfinal 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, 125 mM KC1, 0.2mM EDTA, 0.5mM DTT. 10% DEG). For droplet formation assay with GFP::SRSF2 MEF nuclear extract supplementation. NE was added to lOuM 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. Image series were taken over a 20-minute time span with 1 image every 30 seconds.

[0142] Mass spectrometry to profile condensates composition

[0143] 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 pg 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 w ere dried in a speedvac and resusupended in a solution of 3% acetonitrile and 0.1 % formic acid to a final concentration of 0.5 pg / pL. Mass spectrometry analysis was conducted on a Thermo Fisher QE-HFX coupled to a Vanquish Neo UHPLC. Approximately 1 g of each sample w as loaded onto an EASY-Spray PepMap RSLC C18 column (2 m, 100A, 75 m x 50 cm) and eluted at 300 nl / min over a 120-minute gradient. MSI 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 w as 1.4 m / z and fragmentation w ere 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 le5 and the dynamic exclusion time set to 90s. The collected MS data were analyzed using MSFragger V4.0[l] and searched against the human SwissProt database. The search parameters were set as follows: strict trypsindigestion, 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%.

[0144] 1,6-hexanediol treatment to examine effects of PP on LLPS

[0145] 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 pg / mL) (Gibco). To examine NS LLPS dynamics, EGFP::SC35 MEFs (previously described in 9) were treated with DMSO or 1 pM 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 ImageJ 112 by averaging the tMl and tM2 values.

[0146] 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 (abl56173, Abeam) or MED1 (ab60950, Abeam)) 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 in 9. 20+ cells were measured for each condition.

[0147] Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0148] 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 (3-actin levels were used as controls.

[0149] Chromatin Immunoprecipitation (ChIP)

[0150] ChIP for SC35 was performed using anti-SC35 antibody (abl 1826, Abeam) as previously described 113. 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 pg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 pg 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. ChlP-qPCR for MEFs were essentially performed the same way as previously described with anti-SC35 (abl 1826, Abeam) and anti-XBPls antibody (Biolegend 658802), except that the MEFs were directly fixed with 1% formaldehyde before subject to nuclei isolation and chromatin immunoprecipitation.

[0151] HTS of FDA-approved drugs and quantitative imaging analysis

[0152] EGFP::SC35 MEFs were seeded in black 384 well plate with glass bottom (Cellvis). The FDA-approved compound library (lOOnL 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 pM using a BRAV O 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 in 9.

[0153] 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 XI 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 described 114. 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 described 3. For both doseresponse and Perk promoter-driven dGFP experiments, eight biological replicates were performed per drug per dose.

[0154] mRNA-seq and transcriptome analysis

[0155] 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). Sequencing was performed on a NextSeq 2000 sequencer.

[0156] 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 Trimmomatic 115. Then the processed reads were aligned by HISAT2 116 against mouse reference mmlO. For gene-level intron / exon quantification, bedtools software 117 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 were normalized by gene length and total reads for FPKM normalization.

[0157] The analy sis of drug-treated samples was done using Galaxy 118: Trim Galore was used for quality control and trimming 119 and Salmon 120 was used to normalize thepaired-end reads (TPM method). The online 3D RNA-Seq 121 pipeline was used to determine upregulated and downregulated genes, in addition to generating the PCA plot.

[0158] 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).

[0159] The reads were first mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0122 and the gene expression level was estimated using RSEM vl.2.15123. TMM (trimmed mean of M-values) was used to normalize the gene expression. Differentially expressed genes were identified using the edgeR program!24. Genes showing altered expression with p < 0.05 and more than 1.5-fold changes were considered differentially expressed. Goseq was used to perform the GO enrichment analysis and Kobas was used to perform the pathway analysis.

[0160] References

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[0203] 43. Jafari, R. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nature Protocols 9, 2100-2122 (2014).

[0204] 44. Ilik, I. A. et al. SON and SRRM2 are essential for nuclear speckle formation. eLife 9, e60579 (2020).

[0205] 45. Fei, J. et al. Quantitative analysis of multilayer organization of proteins andRNA in nuclear speckles at super resolution. Journal of Cell Science 130, 4180-4192 (2017).

[0206] 46. Sharma, A., Takata, H., Shibahara, K., Bubulya, A. & Bubulya, P.A. Son is essential for nuclear speckle organization and cell cycle progression. Mol Biol Cell 21, 650-63 (2010).

[0207] 47. Liao, S.E. & Regev, O. Splicing at the phase-separated nuclear speckle interface: a model. Nucleic Acids Res 49, 636-645 (2021).

[0208] 48. Xu, S. et al. SRRM2 organizes splicing condensates to regulate alternative splicing. Nucleic Acids Res 50, 8599-8614 (2022).

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[0210] 50. Sabari, B.R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).

[0211] Additional experimental details of this example can be found as published by bioRxiv, Dian et al., Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1, doi.org / 10. 1101 / 2024.04.18.590103 (April 21, 2024), which is incorporated herein by reference in its entirety .

[0212] Example 2

[0213] Mechanism of action of pyrvinium pamoate (PP)

[0214] Via a cell-free nuclear speckle reconstitution assay, we identified the mechanism of action of PP is by reducing the surface tension of nuclear speckle condensates, via binding to the intrinsically disordered region (IDR) of the nuclear speckle scaffold protein SON. Simply speaking, we identified a mechanism where PP can reduce the surface / interfacial tension of nuclear speckles via targeting SON IDR, leading to larger surface areas withincreased SRSF2 (a subunit of the spliceosome complex) content at the periphery, increased wetting of genomic DNA and subsequently causing a higher portion of spliceosomes stably engaging in active RNA processing and transcription elongation of proteostasis genes (Fig. 7).

[0215] PP rejuvenates nuclear speckles and reduces tau level in human iPSC-neuron models of tauopathy without inducing toxicity' or stress.

[0216] To test if PP reduces Tau level in human neurons, we initially studied V337M 3R-Tau iPSC-neurons (i3N) ’, which unlike 4R-tau-P301S models, do not develop Tau aggregates but models the earlier events of Tau pathophysiology2. IF against nuclear speckle protein SRRM2 revealed that compared to wild-type controls, V337M iPSC-neurons exhibited aberrant nuclear speckle morphology characterized by smaller size and more spherical shape, and 12 hours of lOnM PP treatment fully restored nuclear speckle morphology' to normal size and diffuseness (Fig. 8A)3. Consequently, nanomolar PP treatment boosted the autophagic flux and reduced the level of p-Tau (V337M) within 12h hours (Figs. 8B-8D)3, without inducing cellular stress and toxicity (Figs. 9A, 9B).

[0217] PP reduces tauopathy in flies.

[0218] Pan-neuronal expression of wild-type human MAPT gene in Drosophila recapitulates key features of tauopathies, including hyperphosphorylated tau, age-dependent synaptic and neuron loss, and reduced survival4Thus, we tested the efficacy of PP in ameliorating tauopathy in flies that overexpress human Tau (2N4R) pan-neuronally [e / avc155- Gal4: UAS-hTaul.13 (C155>Tau)] as well as control e / av,cl55-Gal4 (C155) flies5. Both strains of flies were fed with either standard diet or diet supplemented with 25 pM PP, which did not affect the normal development and growth of flies despite its effect in attenuating WNT / Y API signaling6. Tau flies exhibit locomotor impairment at both larval and adult stages. In larval crawling assay and adult fly climbing assay, PP significantly restored motor function in CI55>Tau flies, to a level similar to or even slightly better than control (C 155) flies fed with a standard diet (Figs. 10A, 10B)3. Western blot of the brain lysates of these flies showed a significant reduction of p-Tau, and to a lesser extent, total Tau levels (Fig. 10C)3.

[0219] PP crosses the blood brain barrier and boosts proteostasis gene expression in mice brain.

[0220] For PP to be a viable treatment for neurodegeneration in mammals, it must possess the ability to penetrate the blood-brain barrier. To assess this crucial aspect, we conducted a pharmacokinetics (PK) study in adult male C57BL / 6 mice. Following an intraperitoneal (IP) injection of 0.5mg / kg PP (PP has limited systemic bioavailability when administered orally in humans7), we detected nanomolar range of PP (60ng / ml ~ 0.16pM) in both the plasma and brain as soon as fifteen minutes after IP injection, indicating PP can cross the blood brain barrier (Fig. 11 A). More importantly, both the plasma and brain PP level is close to its EC50 (-100 nM) demonstrated in vitro. To determine whether PP can induce proteostasis gene expression in mice brain in vivo and the optimal time of administration, we IP-injected vehicle or PP (0.5mg / kg) at four different time points (CTO, 6. 18 and 24) and collected hippocampus tissues six hours later. We assayed the expressions of Reep6 and Sei 11 mRNA by qPCR. Reep6 regulates protein trafficking in the ER 8. Sei 11 is indispensable for mammalian ERAD 9. Importantly, knocking out both gene increases Tau aggregates in human iPSC-neurons according to a recent whole-genome CRISPR screen 10. As shown in Fig. 1 IB, PP increased the expression of both genes at all four times, with the highest expression observed when PP is administrated at CTO. Further, examining fold induction of gene expression by PP revealed a ~12h rhythm peaking at CTO and CT 12, likely due to ~12h rhythm of Son expression in the hippocampus (Fig. 1 IB).

[0221] References

[0222] 1 Wang, C. et al. Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening. Stem Cell Reports 9, 1221- 1233 (2017). doi.org: 10. 1016 / j.stemcr.2017.08.019.

[0223] 2 Parra Bravo, C. et al. Human iPSC 4R tauopathy model uncovers modifiers of tau propagation. Cell 187, 2446-2464. e2422 (2024). doi.org: 10.1016 / j. cell.2024.03.015.

[0224] 3 Dion, W. et al. Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1. bioRxiv, 2024.2004.2018.590103 (2024). doi.org: 10. 1101 / 2024.04. 18.590103.

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Claims

WHAT IS CLAIMED IS:

1. A method of reducing tauopathy comprising administering to a subject in need thereof an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the method reduces Tau levels of the subject.

3. The method of claim 2, wherein the method reduces hyperphosphorylated Tau levels of the subject.

4. The method of claim 2, wherein the method reduces total Tau levels of the subject.

5. The method of claim 1, wherein the pyrvinium compound is administered in a pharmaceutical composition further comprising at least one pharmaceutical carrier.

6. The method of claim 1, wherein the subject is a human patient.

7. The method of claim 1, wherein the pyrvinium compound is pyrvinium pamoate.

8. The method of claim 7, wherein the pyrvinium pamoate is administered in a dosage of from 0.04mg / kg to 5mg / kg.

9. The method of claim 1, wherein the subject suffers from a disease selected from proteinopathy, Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), retinitis pigmentosa (RP), retinitis pigmentosa (RP) with rhodopsin mutations, aging, neurodegenerative diseases, and metabolic disorders.

10. The method of claim 1, wherein the method exhibits one or more or all of modulating global proteostasis, enhancing protein quality control, suppressing YAP 1 signaling, enhancing the propensity of nuclear speckle liquid-liquid phase separation (LLPS), improving Son gene expression, reducing the surface tension and / or increasing the surface areas of nuclear speckles, upregulating protein quality’ control gene expression, downregulating YAP1 transcription output, reducing tauopathy, promoting autophagy, promoting ubiquitin-proteasome system (UPS) activity, and preventing retina degeneration.

11. A method of treating a disease associated with accumulation of misfolded proteins, the method comprising administering to a subject in need thereof an effective amount of a pyrvinium compound, or a pharmaceutically acceptable salt thereof.

12. The method of claim 11, wherein the disease is selected from proteinopathy, Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), retinitis pigmentosa (RP), retinitis pigmentosa (RP) with rhodopsin mutations, aging, neurodegenerative diseases, and metabolic disorders.

13. The method of claim 11 , wherein the method exhibits one or more or all of: modulating global proteostasis, enhancing protein quality control, suppressing YAP 1 signaling, enhancing the propensity7of nuclear speckle liquid-liquid phase separation (LLPS), improving Son gene expression, reducing the surface tension and / or increasing the surface areas of nuclear speckles, upregulating protein quality7control gene expression, downregulating YAP1 transcription output, reducing tauopathy, promoting autophagy, promoting ubiquitin-proteasome system (UPS) activity7, and preventing retina degeneration.

14. The method of claim 1 1. wherein the pyrvinium compound is administered in a pharmaceutical composition further comprising.

15. The method of claim 11, wherein the subject is a human patient.1 . The method of claim 11, wherein the pyrvinium compound is pyrvinium pamoate.

17. The method of claim 11. wherein the pyrvinium pamoate is administered in a dosage of from 0.04mg / kg to 5mg / kg.

18. A pharmaceutical composition comprising at least one pharmaceutical carrier and pyrvinium pamoate, or a pharmaceutically acceptable salt thereof, in a dosage of from 0.04mg / kg to 5mg / kg.

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