Protective molecules and therapeutic targets for tauopathy

Abstract

This document relates to materials and methods for treating a tauopathy. For example, this document provides methods of treatment of a tauopathy that include administering a pharmaceutical preparation comprising a pharmaceutically effective amount of a compound selected from the group of staurosporine, midostaurin, and biologically active analogs thereof.

Description

[0001] Attorney Docket No.: 49045-0085WO1 / 05762 PROTECTIVE MOLECULES AND THERAPEUTIC TARGETS FOR TAUOPATHY CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the filing date of U.S. Provisional Patent Application No.63 / 518,592, filed on August 10, 2023. The contents of U.S. Provisional Patent Application No.63 / 518,592 are incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERAL FUNDING This invention was made with government support under grant numbers BX003168-05 and BX003168-01 awarded by the US Department of Veterans Affair and grant no. NS080881 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named “49045-0085WO1_SL.xml.” The XML file, created on August 2, 2024, is 24,203 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. BACKGROUND 1. Technical Field This document relates to methods and materials for treating a tauopathy (e.g., progressive supranuclear palsy). For example, this document provides methods and materials for using staurosporine, midostaurin, and / or biologically active analogs thereof to treat a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). In some cases, a method for treating a tauopathy (e.g., progressive supranuclear palsy) within a mammal (e.g., a human) can include administering a composition comprising a pharmaceutically effective amount of a compound selected from the group of consisting of staurosporine, midostaurin, and biologically active analogs thereof. This document also provides methods and materials for identifying an agent as having the ability to improve a neurological function within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). Attorney Docket No.: 49045-0085WO1 / 05762 2. Background Information The following information is provided to assist the reader in understanding technologies disclosed below and the environment in which such technologies may typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise in this document. References set forth herein may facilitate understanding of the technologies or the background thereof. The disclosure of all references cited herein are incorporated by reference. Tauopathies are a group of common and devastating neurodegenerative diseases, including Alzheimer’s disease, chronic traumatic encephalopathy, progressive supranuclear palsy and several other types of frontotemporal dementia. There is no effective treatment for any of these conditions, and the discovery of effective therapeutics is expected to have significant impact on the field and on clinical practice. Tauopathies are characterized pathologically by neuronal loss, accumulation of aggregates of the microtubule-associated protein Tau in surviving CNS neurons, and microglial infiltration and activation. Collectively, these diseases, which include progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE) and Alzheimer’s disease (AD), are common and important causes of chronic neurological disabilities in numerous people. Current treatments do not alter the course of these diseases; as a result, there is an urgent unmet need for new approaches that address pathogenic mechanisms and prevent progression. Accumulating evidence links CNS innate immunity directly to the pathogenesis of tauopathy. In particular, compelling data show that complement-dependent synaptic pruning mediated by microglia is an early and possibly preventable event underlying the neurological deficits of tauopathy. The microtubule-associated protein Tau was initially identified as a factor that promotes assembly of tubulin into microtubules. The microtubule-binding domain of Tau localizes to the C-terminal half of the protein. The N-terminal domain contains a proline-rich region and is thought to be involved in interactions with other cellular components. Tau is expressed widely in CNS neurons, and is enriched in the axonal compartment, where it functions in microtubule stabilization and regulation of fast axonal transport. Tau is encoded by the MAPT gene. Alternative splicing in the adult human brain produces six protein isoforms. Depending on the presence of exon 10 in the mRNA, Tau contains either three or four copies of a repeat containing tubulin-binding Attorney Docket No.: 49045-0085WO1 / 05762 sequences, referred to as 3R- and 4R-Tau respectively. Greater than 40 Tau phosphorylation sites have been identified, mostly flanking the tubulin-binding domain. Tau is pathologically hyperphosphorylated in tauopathy. Phosphorylation promotes Tau detachment from microtubules, suggesting it regulates microtubule stability. Specific Tau isoforms accumulate different tauopathies: in AD, all six Tau isoforms are present, whereas PSP lesions are mainly composed of 4-repeat isoforms (particularly 4R / 0N). The pathogenic role of Tau in tauopathies is strongly supported by genetic evidence. There is a strong association between PSP and homozygosity for the H1 haplotype at the MAPT locus (the H1 haplotype produces disproportionately more exon 10+ MAPT mRNA) and genome-wide association studies showed a robust association between single nucleotide polymorphisms at the MAPT locus and risk of PSP. Furthermore, a Mendelian neurodegenerative disorder – frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP17) – is caused by mutations in the MAPT gene. The discovery of pathogenic MAPT mutations provided important confirmation that abnormalities of Tau are a primary cause of neurodegeneration. The close clinical and pathologic similarity between FTDP17 and sporadic tauopathies suggests that abnormal functions of Tau are central to neurodegeneration in common sporadic tauopathies. Increasing evidence implicates innate immunity in the pathophysiology of tauopathy. Microgliosis and synapse loss were amongst the earliest pathological changes in mice expressing human P301S mutant Tau. Immunosuppression with FK506, or microglial depletion using a CSF1R inhibitor, mitigated neuroinflammation, synapse loss and other phenotypic endpoints in this model. Microglia play an important role in developmental CNS synaptic pruning responding to deposition of complement C1 and C3 to eliminate inactive synapses. Complement is upregulated in human tauopathy brain and recent work suggests that the developmental synaptic pruning pathway is reactivated in neurodegenerative disease. Thus, C1q deposition at CNS synapses, and complement- dependent microglial infiltration and synaptic loss were early events in mice expressing mutant APP, APP + PS1 or Tau. Neuronal oxidative stress is a prominent finding in tauopathy, although its origin is controversial. Microglial activation is associated with formation of reactive oxygen species: pro-inflammatory signaling promotes assembly of membrane NADPH oxidase, resulting in generation of superoxide. Formation of hydrogen peroxide is then catalyzed by superoxide dismutase (SOD2), which is expressed strongly Attorney Docket No.: 49045-0085WO1 / 05762 in activated microglia. Peroxide is freely diffusible into adjacent neuronal cells, where it may contribute to neuropathology. Although microglial elimination of dead cells and protein aggregates is known to occur in neurodegeneration and is presumed to be a compensatory response that mitigates deleterious effects of cellular debris and amyloid fibrils, recent work suggests that microglial engulfment of protein aggregates may in fact allow spreading of seeding-competent aggregate species through the CNS. These considerations strongly indicate that neuroinflammation contributes significantly to the neurological deficits observed in tauopathy, presenting important, novel molecular targets for therapeutic intervention. Zebrafish have been increasingly employed as an animal model to study the pathogenesis of human neurological diseases. Zebrafish larvae can be manipulated to be optically transparent, enabling direct visualization of neurons, neural circuitry formation and neuronal activity in vivo, by expression of transgenes encoding reporter proteins. Zebrafish breed regularly and produce large clutches of offspring, which can be housed practicably. This has allowed phenotype-based genetic and small molecules screens to isolate novel mutants and chemicals that modify disease-relevant phenotypes in vivo. Three parallel lines of evidence have firmly established zebrafish as highly relevant models to understanding human disease. First, extensive phylogenetic conservation of key genes implicated in human neurological diseases, and functional substitution of zebrafish genes by the human homologue indicate that relevant molecular mechanisms are conserved phylogenetically. Second, cellular populations analogous to those involved in human diseases are present in the zebrafish CNS; as a result, cell type-specific and cell non-autonomous pathologies that occur in human neurological diseases can be recapitulated in the zebrafish nervous system. Third, conservation of vertebrate CNS organization, neurochemical systems and circuits allows the role of specific neuronal groups and neurotransmitters to be investigated in particular types of behavioral abnormalities. Zebrafish microglia show similar morphology, ontogeny, transcriptional programs, and phagocytic properties to microglia in other vertebrates. Because zebrafish can be engineered to be transparent, transgenic zebrafish lines expressing fluorescent reporters in microglia allow direct intravital imaging of microglial morphology and dynamics. Other aspects of innate immunity in zebrafish including the complement system are highly conserved with respect to mammals and other higher vertebrates. Attorney Docket No.: 49045-0085WO1 / 05762 SUMMARY This document provides methods and materials for treating a tauopathy (e.g., progressive supranuclear palsy). For example, this document provides methods and materials for using staurosporine, midostaurin, and / or biologically active analogs thereof to treat a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). In some cases, a method for treating a tauopathy (e.g., progressive supranuclear palsy) within a mammal (e.g., a human) can include administering a composition comprising a pharmaceutically effective amount of a compound selected from the group of consisting of staurosporine, midostaurin, and biologically active analogs thereof. As described herein, staurosporine, midostaurin, and biologically active analogs thereof can be used to improve one or more neurological functions within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). Having the ability to use staurosporine, midostaurin, and / or biologically active analogs thereof to improve one or more neurological functions within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy) provides patients and clinicians with treatment options for treating a tauopathy. This document also provides methods and materials for identifying an agent as having the ability to improve a neurological function within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). As described herein, engineered zebrafish can be used to identify agents as having the ability to improve a neurological function within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy). Having the ability to use identify additional agents as having the ability to improve a neurological function within a mammal (e.g., a human) having a tauopathy (e.g., progressive supranuclear palsy) provides patients and clinicians with additional treatment options for treating a tauopathy such as progressive supranuclear palsy. In one aspect, this document features a method of treatment of tauopathy that includes administering a pharmaceutical preparation comprising a pharmaceutically effective amount of a compound selected from the group of staurosporine, midostaurin, and biologically active analogs thereof. In a number of embodiments, the compound is midostaurin or a biologically active analog thereof. The tauopathy may, for example, be progressive supranuclear palsy. In another aspect, this document features a method of screening for activity in Attorney Docket No.: 49045-0085WO1 / 05762 treatment of tauopathy that includes selecting one or more agents to screen, for each one of the one or more agents, exposing one or more transgenic zebrafish expressing human Tau to the (selected) one of the one or more agents, and comparing neurological function of the transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents to neurological function of other zebrafish expressing human Tau which were not exposed to the one of the one or more agents. The human Tau protein can be wildtype (WT) or mutant human Tau, including isoforms thereof. In a number of embodiments, the transgenic zebrafish expressing human Tau express wildtype or non- mutant human Tau. In a number of embodiments, the transgenic zebrafish express human 4R-Tau. The transgenic zebrafish may, for example, express human 0N / 4R-Tau or human P301L 0N / 4R-Tau. In a number of embodiments, the method includes, for each one of the one or more agents, exposing a group of transgenic zebrafish expressing human Tau to the one of the one or more agents, and comparing neurological function of the group of transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents to neurological function of a group of transgenic zebrafish expressing human Tau and which were not exposed to the one of the one or more agents. In a number of embodiments, comparing neurological function of the transgenic zebrafish expressing human Tau and exposed to the one of the one or more agent to the neurological function of the transgenic zebrafish expressing human Tau and which were not exposed to the one of the one or more agents includes comparison thereof to neurological function of one or more control zebrafish not expressing human Tau. In a number of embodiments, one or more neurological functions of a plurality of transgenic zebrafish expressing human Tau within the group of transgenic zebrafish expressing human Tau are compared to the one or more neurological functions of a plurality of transgenic zebrafish expressing human Tau and which were not exposed to the one of the one or more agents. The method may further include quantifying the one or more neurological functions and comparing a mean (and / or other statistical characterization) of each of the one or more neurological functions of the plurality of transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents with a corresponding mean (and / or other statistical characterization) of the one or more neurological functions of the plurality of transgenic zebrafish expressing human Tau and which were not exposed to the one of the one or more agents. The one or more Attorney Docket No.: 49045-0085WO1 / 05762 neurological functions may, for example, be selected from swimming motion and eye motion. The one or more neurological functions may, for example, be quantified via software-based analysis of videographic data. In a number of embodiments, the one or more agents to screen are selected from at least one of Brd4 inhibitors, histone deacetylases inhibitors, DNA methyltransferase inhibitors, or kinase inhibitors. The one or more agents to screen may, for example, be selected from at least one of known Brd4 inhibitors or kinase inhibitors. In a number of embodiments, the transgenic zebrafish expressing human Tau may include one or more transgene alleles include an encoded GAL4 driver and a UAS responder cassette including encoded wild type human Tau (for example, wild-type human Tau), an encoded imaging reporter, and a self-cleaving peptide separating the encoded human Tau and the encoded imaging reporter. In a number of embodiments, the transgenic zebrafish expressing human Tau include one or more transgene alleles including an encoded GAL4 driver and UAS responder cassette including encoded wild type human 0N / 4R-Tau, an encoded imaging reporter, and a self-cleaving peptide separating the encoded wild type human 0N / 4R-Tau and the encoded imaging reporter. The encoded imaging reporter may, for example, be a fluorescent protein. The encoded imaging reporter may, for example, be mCherry. In some cases, a targeting signal may be associated with the encoded imaging reporter so that the human Tau and the imaging reporter are expressed independently in separate tissue compartments. In a number of embodiments, the targeting signal is a nuclear localization signal (nls). In a number of embodiments, the one or more agents are one or more chemical compounds. In a number of embodiments, the method is a high throughput screening. The tauopathy may, for example, be progressive supranuclear palsy. In another aspect, this document features a method for treating a mammal having a tauopathy. The method comprises (or consists essentially of or consists of) administering, to the mammal, a composition comprising a pharmaceutically effective amount of a compound selected from the group consisting of staurosporine, midostaurin, and biologically active analogs thereof. The mammal can be a human. The compound can be midostaurin. The tauopathy can be progressive supranuclear palsy. The method can comprise identifying the mammal as having the tauopathy prior to the administering step. Attorney Docket No.: 49045-0085WO1 / 05762 In another aspect, this document features a method for identifying an agent for treating a tauopathy within a mammal. The method comprises (or consists essentially of or consists of) (a) exposing one or more transgenic zebrafish expressing a human Tau to a test agent; and (b) determining the presence of an increased level of a neurological function of the one or more transgenic zebrafish expressing the human Tau and exposed to the test agent as compared to the level of the neurological function of one or more zebrafish expressing the human Tau that were not exposed to the test agent, thereby identifying the test agent as being the agent for treating the tauopathy within the mammal. The transgenic zebrafish expressing the human Tau can express wildtype or non-mutant human Tau. The transgenic zebrafish expressing the human Tau can express 4R-Tau. The transgenic zebrafish expressing the human Tau can express 0N / 4R-Tau or human P301L 0N / 4R-Tau. The neurological function can be selected from the group consisting of swimming motion and eye motion. The test agent can be selected from the group consisting of Brd4 inhibitors, histone deacetylases inhibitors, DNA methyltransferase inhibitors, and kinase inhibitors. The neurological function can be quantified via software-based analysis of videographic data. The transgenic zebrafish expressing the human Tau can comprise one or more transgene alleles comprising an encoded GAL4 driver and a UAS responder cassette comprising encoded human Tau, an encoded imaging reporter, and a self-cleaving peptide separating the encoded human Tau and the encoded imaging reporter. The transgenic zebrafish expressing the human Tau can comprise one or more transgene alleles comprising an encoded GAL4 driver and UAS responder cassette comprising encoded wild type human 0N / 4R-Tau, an encoded imaging reporter, and a self-cleaving peptide separating the encoded wild type human 0N / 4R-Tau and the encoded imaging reporter. The encoded imaging reporter can be a fluorescent protein. The encoded imaging reporter can be mCherry. A targeting signal can be associated with the encoded imaging reporter so that the human Tau and the imaging reporter are expressed independently in separate tissue compartments. The targeting signal can be a nuclear localization signal (nls). The tauopathy can be progressive supranuclear palsy. The mammal can be a human. In another aspect, this document features methods for treating a mammal (e.g., a human) having a tauopathy. The method comprises (or consists essentially of or consists of) administering, to said mammal, a composition including from about 50 mg to about 100 mg of midostaurin. The human can be a mammal. The composition can be Attorney Docket No.: 49045-0085WO1 / 05762 administered from 1 to about four times a day. The composition can be administered twice a day. The tauopathy can be progressive supranuclear palsy. The present compositions, systems, methods, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. DESCRIPTION OF DRAWINGS Figure 1 illustrates summary data of a screen of 147 modulators of epigenetic readers, writers, and erasers for rescue of hypokinesia in Tau zebrafish. Tau zebrafish were exposed to compounds from 2dpf – 5dpf then % rescue of mean speed in the light phase of the VMR was calculated. Each data point shows % rescue ± SE for each library compound, ordered by rank along the x-axis. The untreated Tau (0% rescue) and Sib (100% rescue) groups are shown for reference. Limits showing the a priori definition of a ‘hit’ (library mean rescue ± 3 SD) are indicated. Three compounds satisfying this definition are shaded (and labelled as 1, 2, and 3) and their properties detailed in Table 1. For (+)JQ1, the inactive stereoisomer (í)JQ1 is labelled for comparison. Figure 2 illustrates summary data of a screen of 80 inhibitors of kinases for rescue of hypokinesia in Tau zebrafish. Tau zebrafish were exposed to compounds from 2dpf – 5dpf then % rescue of mean speed in the light phase of the VMR was calculated. Each data point shows % rescue ± SE for each library compound, ordered by rank along the x- axis. The untreated Tau (0% rescue) and Sib (100% rescue) groups are shown for reference. Limits showing the a priori definition of a ‘hit’ (library mean rescue ± 3 SD) Attorney Docket No.: 49045-0085WO1 / 05762 are indicated. Two compounds satisfying this definition are shaded (and labelled 5 and 4) and their properties detailed in Table 2. Figure 3 illustrates rescue of the motor phenotype in Tauopathy zebrafish by exposure to midostaurin. Panel A: Verification of activity of midostaurin. Graphs show mean swimming speed over time. The light traces show mean displacement of zebrafish at each video frame transition in the recording, scaled to show speed. The dark traces show the same data averaged over a moving 10-second window to better clarify the data trend. Panel B: Individual mean swimming speeds for 72 zebrafish in each group combined from three independent assays, illustrating the dose-dependent quantitative rescue of mean swimming speed by midostaurin in Tau zebrafish. Data points show individual zebrafish, bars show mean ± 95% CI. Figure 4 illustrates in Panel A example traces of ocular angle against time for control, Tau zebrafish, and Tau zebrafish exposed to midostaurin. Panel B illustrates ocular range of movement for 15-20 zebrafish in each group combined from three independent assays. Bars show mean ± 95% CI. Panel C illustrates reflex gain for 15-20 zebrafish in each group combined from three independent assays. Bars show mean ± 95% CI. Panel D illustrates saccade frequency for 15-20 zebrafish in each group combined from three independent assays. In each of Panels B-D, datapoints show individual zebrafish, and bars show mean ± 95% CI. Figure 5 illustrates chemical structures of (+)-JQ1 (Compound #1; a thienotriazolodiazepine), trichostatin A (Compound #2), 2,4-DPD (2,4- pyridinedicarboxylic acid, diethyl ester; Compound #3), staurosporine (Compound #4), and midostaurin (Compound #5). Figure 6 illustrates transgenic zebrafish expressing human 4R / 0N-Tau conditionally in neurons show impaired survival, neurodegeneration and microgliosis. Panel A: Expression of human 0N / 4R-Tau conditionally in neurons of transgenic Tau zebrafish using Gal4 / UAS genetics. Panel B: Cytoplasmic expression of human 4R-Tau and nuclear expression of mCherry (to allow rapid non-invasive genotyping) as separate proteins from the same bicistronic hsa. MAPT-2a-nls-mCherry mRNA. Panel C: Survival curves of Tau zebrafish in comparison with non-expressing siblings (Non-Tg), and Ctrl zebrafish expressing nls-mCherry only. ****p<0.0001 Tau vs. Ctrl or Tau vs. Non-Tg, Mantel-Cox test. Panel D: 15-day survival of three biological replicate cohorts each containing Tau, Ctrl and Non-Tg sibling controls. Bars show mean ± SE, points show % Attorney Docket No.: 49045-0085WO1 / 05762 survival for each replicate. ****p<0.00011-way ANOVA with Dunnett’s multiple comparisons test. Panels E – G: Three complementary methods demonstrating cell death in the CNS of Tau and Ctrl zebrafish from 2 – 7 days post-fertilization (dpf): (Panel E) acridine orange labeling and intravital imaging of spinal cord; (Panel F) TUNEL labeling of brain sections; (Panel G) cleaved caspase 3 labeling of brain sections. Example images from each method are shown above, and quantification shown below; data points show individual zebrafish (Ctrl, blue circles; Tau, red triangles), bars show group mean ± SE. p<0.05*, 0.01**, 0.001***, 0.0001*** Ctrl vs. Tau, 2-way ANOVA (genotype, with Šidák test. Inset panel in Panel G shows western at 3dpf probed with an antibody specific to cleaved (activated) caspase-3, and ȕ-actin as a loading control. Panel H: Western blots of lysates from Tau and Ctrl larvae at 5dpf probed with antibodies to tyrosine hydroxylase (TH; dopaminergic neurons), glutamic acid decarboxylase (GAD; GABAergic neurons), synaptophysin (SYP; presynaptic terminals) or post-synaptic density protein 95 (PSD95, postsynaptic terminals) and ȕ-actin (loading control). Example blots are shown above, quantification of expression of each marker in Tau zebrafish (red triangles) relative to Ctrl (blue circles) is shown in four biological replicates below. Bars show mean ± SE, p<0.01** Ctrl vs. Tau, 2-tailed t-test with Welch’s correction. Panel I omitted. Panel J: Microgliosis in Tau zebrafish. Brain sections were labeled with antibody 7.4.C4 (a microglial marker; example images on left). Brain microglia were counted in serial sections from larvae at time points 2 – 7 dpf and counts normalized to section area. Points show mean for 10 – 12 sections of individual Tau (red triangles) and Ctrl (blue circles) larvae, bars show group mean ± SE, p<0.05*, 0.0001*** Ctrl vs. Tau, 2-way ANOVA (genotype, timepoint) with Šidák multiple comparisons test. Figure 7 illustrates human 4R / 0N-Tau becomes hyperphosphorylated, mislocalized, misfolded, truncated, oligomerized and insoluble in the CNS of transgenic zebrafish. Panels A, B: Western blots of zebrafish head region lysates. (Panel A) Samples from Ctrl and Tau zebrafish, blot probed with a polyclonal antibody to total human Tau and ȕ-Actin as a loading control. (Panel B) Lysates from Tau zebrafish were pretreated with calf intestinal alkaline phosphatase (CIP), Lambda protein phosphatase (^PP), or no enzyme (í). The blot was probed with an antibody to total human Tau (left) and an antibody to human phosphorylated [pS202, pT205]-Tau (AT8; right). Panel C: Replicate western blots of lysates from Ctrl and Tau zebrafish were probed with a panel of Attorney Docket No.: 49045-0085WO1 / 05762 antibodies specific to different phosphorylated human Tau epitopes. The Ponceau-S loading control is shown for the first blot (left; others identical). Panel D: Major domains of human 4R / 0N-Tau are shown to illustrate the locations of epitopes detected by the antibodies used in this study. Panel E: Serial axial sections (planes indicated in schematic at top) through a Tau zebrafish at 5dpf labeled for human phosphorylated Tau (AT8; green) and a nuclear counter label (DAPI; blue). The inset panels top left show similar sections from Ctrl and Tau zebrafish at higher magnification. The inset panels below right show sections labeled with antibodies to other human Tau phosphoepitopes and misfolding epitopes as indicated. Panel F: Confocal micrographs showing sections from Tau and Ctrl brain labeled with antibodies recognizing misfolded human Tau (Alz50 and MC1; green) and a nuclear counter label (DAPI; blue). Panel G: Confocal micrographs showing sections from Tau and Ctrl brain labeled with an antibody recognizing human Tau truncated at D421 (TauC3; green) and a nuclear counter label (DAPI; blue). Panel H: Western blots of head region lysates from Ctrl zebrafish at 5dpf and Tau zebrafish at 3 – 6 dpf that were extracted with RIPA (detergent-soluble proteins; left) or DIGE (detergent insoluble proteins; right) buffers. Blots were probed with antibodies to total human Tau (top), phosphorylated human Tau (AT8; middle) and ȕ-Actin (loading control; bottom). Panel I omitted. Panel J: Blue native gel western blot of head region lysates from Ctrl and Tau zebrafish probed with an antibody to total human Tau. The expected electrophoretic mobility of monomeric human Tau is indicated by an arrow; the asterisks show additional high molecular weight forms. Figure 8 illustrates neurological phenotypes resembling progressive supranuclear palsy in transgenic zebrafish expressing human 4R / 0N-Tau. Panels A – F: Zebrafish motor function was evaluated at 5dpf using a well-validated 96-well plate automated tracking system1,2under constant ambient illumination at 28°C. (Panel A) Single video frame illustrating the plate image. The inset shows features detected by the algorithm in a single well (well boundary, red; zebrafish, blue; the calculated zebrafish centroid position is indicated by a yellow marker). (Panel B) 48 Ctrl zebrafish were compared with 48 Tau zebrafish in the same 96-well plate. The graph shows zebrafish centroid displacement at each video frame transition scaled to show speed (y-axis) over 60 minutes of stable recording (x-axis). The light gray traces show the group means at each frame transition. The colored markers (Ctrl, blue; Tau, red) show group mean ± SE within each sequential 2-minute time bin. ****p<0.0001, Ctrl vs. Tau, 2-way repeated measures ANOVA with Attorney Docket No.: 49045-0085WO1 / 05762 Šidák post hoc test. (Panels C – E) Quantification of motor function in Ctrl and Tau zebrafish from three combined biological replicate experiments: (Panel C) mean scalar speed (total displacement during assay / duration of assay); (Panel D) % time active (% of video frame transitions showing zebrafish centroid displacement over the course of the assay); (Panel E) inter-movement interval (mean time interval between each centroid displacement event). In each graph, data points show individual zebrafish, bars show mean ± SE. ****p<10í15, Ctrl vs. Tau, 2-tailed unpaired t-test with Welch’s correction for unequal variance. (Panel F) Total displacement at different instantaneous swimming speeds for Ctrl and Tau zebrafish. Panels G – H: Zebrafish swimming kinematics were evaluated at 5dpf in response to abrupt ambient light-dark transitions using high-speed macrovideography and a well-validated segmentation / kinematics application3. (Panel G) Stereotypical ‘O’-band illustrated by superimposing the pseudocolored zebrafish silhouette every 10 ms during the response according to the color scale shown. (Panel H) Peak truncal curvature during ‘O’-bend response for Ctrl and Tau zebrafish. Data points show individual zebrafish, bars show mean ± SE. ns, not significant, Ctrl vs. Tau, 2-tailed unpaired t-test with Welch’s correction for unequal variance. Panel I omitted. Panels J – N: Optokinetic reflexes were elicited at 5pdf by exposing the entire left visual field of the zebrafish to a 15° / cycle grating pattern projected onto a curved screen and animated to rotate in a nasotemporal or temporonasal direction at 15° / s. Ocular movements were recorded by infrared videomicrography and detected, measured, and analyzed using a well-validated automated tracking system4. (Panel J) Single video frames showing the extremes of ocular position after tracking a nasotemporal (above) ortemporonasal (below) stimulus. The ocular axes detected by the software are shown in white. (Panel K) Example nystagmograms showing ocular angle (0 denotes long axis of eye parallel to y- axis of image) for the left (stimulated; solid line) and right (contralateral; broken line) eyes from Ctrl (blue; above) and Tau (red; below) zebrafish in response to successive 1- minute periods of alternating (10s each direction), nasotemporal or temporonasal stimuli. (Panels L – N) Quantification of OKRs in Ctrl and Tau zebrafish: (L) ocular range (difference between maximum and minimum angles in stimulated eye); (Panel M) reflex gain (angular velocity of stimulated eye / angular velocity of stimulus); (Panel N) saccade frequency (frequency of rapid resetting movements in opposite direction to stimulus). In each graph, data points show individual zebrafish, bars show mean ± SE. ****p<0.0001, Attorney Docket No.: 49045-0085WO1 / 05762 Ctrl vs. Tau, 2-tailed unpaired t-test with Welch’s correction for unequal variance. References for Figure 8 are: 1. Zhou, Y., Cattley, R.T., Cario, C.L., Bai, Q. & Burton, E.A. Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications. Nat Protoc 9, 1533-48 (2014). 2. Farrell, T.C. et al. Evaluation of spontaneous propulsive movement as a screening tool to detect rescue of Parkinsonism phenotypes in zebrafish models. Neurobiol Dis 44, 9-18 (2011). 3. Hossainian, D. et al. Quantification of functional recovery in a larval zebrafish model of spinal cord injury. J Neurosci Res 100, 2044-2054 (2022). 4. Scheetz, S.D. et al. An open-source method to analyze optokinetic reflex responses in larval zebrafish. J Neurosci Methods 293, 329-337 (2018). Figure 9 illustrates a chemical modifier screen to identify inhibitors of epigenetic readers, writer and erasers that rescue hypokinesia in transgenic zebrafish expressing human 4R / 0N-Tau. Panel A: Quantification of mean speed (total displacement during assay / duration of assay) during the light phase of the visual motor response in Tau zebrafish and their non-expressing siblings. Data points show individual zebrafish on the left graph and averages for groups of 12 zebrafish on the right, bars show mean ± SE on both graphs. The Z-factor for the assay comparing Tau and Sib zebrafish is shown for both individual zebrafish and averaged groups. Panel B: Z-factor was calculated for the same primary data shown in panel A, either by single zebrafish, or by groups containing 2 – 16 zebrafish each in 100 different random groupings. Panel C: Design of screening assay with 8 groups of 12 zebrafish in each 96-well plate. Untreated Sib and Tau groups in each assay provide controls that define assay QC criteria and allow calculation of % rescue for each of 6 groups of Tau zebrafish exposed to chemicals. Panel D: Summary data of a screen of 140 inhibitors of epigenetic readers, writers, and erasers for rescue of hypokinesia in Tau zebrafish. Tau zebrafish were exposed to chemical from 2dpf – 5dpf then % rescue of mean speed in the light phase of the VMR calculated as shown in panel C. Each data point shows % rescue ± SE for each library compound, ordered by rank along the x-axis (numerical data, chemical identities, and targets shown in Table 10). The untreated Tau (0% rescue) and Sib (100% rescue) groups are shown for reference. Limits showing the a priori definition of a ‘hit’ (library mean rescue ± 3 SD) are indicated. Three compounds satisfying this definition are shaded yellow and their properties detailed in the inset table D’. For (+)JQ1 the inactive stereoisomer (í)JQ1 is shaded green for comparison. Panel E: Data from panel D grouped by biological target. Bars Attorney Docket No.: 49045-0085WO1 / 05762 show group mean ± SE, *p<0.05 group mean vs. rescue = 0, 1-sample t-test. Targets showing significant rescue across the compounds within a group are shaded green (numerical data in Table 11). Abbreviations: BET, bromo- and extra-terminal domain containing proteins; HDAC, histone deacetylases; DNA-MT, DNA methyltransferases; HDM, histone demethylases; HAT, histone acetyltransferases; HMT, histone methyltransferases; Other, various targets listed in Table 12. Panel F: Repurchased (+)JQ1 was tested in Tau zebrafish (green) in comparison with untreated sibling Tau zebrafish (red) and non-transgenic siblings (black) to verify the identity of hit #1. Example 1-minute swim vectors are shown above; graphs below show mean speed during the light phase of the VMR. Gray trace shows mean frame-to-frame displacement scaled to speed; colored markers and bars show 1-minute binned group mean ± SE, similar to Figure 8, panel B. Figure 10 illustrates (+)JQ1 rescues hypokinesia in transgenic zebrafish expressing human 4R / 0N-Tau by preventing microglial synaptic pruning. Panels A – C: Mean speed of Ctrl and Tau zebrafish during the light phase of the VMR at 5dpf following exposure to chemical inhibitors as indicated from 2 – 5dpf. Data points show individual zebrafish, bars show mean ± SE. (Panel A) Tau zebrafish exposed to incremental concentrations of (+)JQ1; (Panel B) Tau zebrafish exposed to incremental concentrations of (í)JQ1; (Panel C) Ctrl zebrafish exposed to incremental concentrations of (+)JQ1; untreated Tau and / or Ctrl zebrafish are shown for comparison in each graph. p<0.0001****, 0.01**, 1-way ANOVA with Dunnett’s multiple comparisons test. Panel D: Western blot showing expression of total human Tau (above) and ȕ-Actin (loading control; below) in Ctrl and Tau zebrafish at 5dpf after treatment with (+)JQ1 or no chemical 2 – 5dpf. Quantification of relative Tau expression in 5 biological replicate experiments is shown below. p<0.0001****, 1-way ANOVA with Dunnett’s multiple comparisons test. Panels E – F: mCherry expression by confocal intravital imaging at 5dpf in Tau zebrafish exposed to (+)JQ1 or no chemical as indicated. (Panel E) Example maximum intensity projection images. (Panel F) Quantification of fluorescence signal in 9 zebrafish per group, data points show individual zebrafish, bars show mean ± SE, data analyzed by 2-way unpaired t-test. Panels G – J: Survival of Ctrl and Tau zebrafish exposed to (+)JQ1 or no chemical. (Panel G) Example survival curves, ****p<0.0001 Tau vs. Tau + (+)JQ1, Mantel-Cox test. (H) 15-day survival of three biological replicate cohorts, each identical to panel G. Panel I omitted. (Panel J) Median survival of Tau Attorney Docket No.: 49045-0085WO1 / 05762 zebrafish exposed to (+)JQ1 or no chemical, from the three biological replicate cohorts shown in H. Points in Panels G and H show % survival for each replicate, bars show mean ± SE. ****p<0.00011-way ANOVA with Šidák multiple comparisons test. Panel K: Acridine Orange labeled spinal cord cells were quantified in Ctrl and Tau zebrafish exposed to no chemical, (+)JQ1, or (í)JQ1, as shown in Figure 6, panel E. Data points show individual zebrafish, bars show mean ± SE. ****p<0.00011-way ANOVA with Šidák multiple comparisons test. Panels L, M: Microglia were labeled in axial sections of 5dpf Tau zebrafish exposed to no chemical or (+)JQ1 using 7.4.C4 antibody and an immunohistochemical reaction, then quantified as shown in Figure 6, panel J. (Panel L) Example images from Tau (above) and Tau + (+)JQ1 zebrafish (below). (Panel M) Microglial quantification; data points show mean values for 10 – 12 sections from individual zebrafish, bars show group mean ± SE. p<0.0001****, 1-way ANOVA with Šidák multiple comparisons test. Panel N: Western blot showing expression of PSD95 (above) and ȕ-Actin (loading control; below) in head region lysates from Ctrl and Tau zebrafish at 5dpf after treatment with (+)JQ1 or no chemical between 2 – 5dpf. Quantification of relative PSD95 expression in 8 biological replicate experiments is shown below. p<0.0001****, 1-way ANOVA with Dunnett’s multiple comparisons test. Panel O omitted. Panel P: Whole mount immunofluorescence for PSD95-immunoreactive post-synaptic puncta in the telencephalon (above) or optic tectum (below; approximate regions of images shown in low-magnification panel to left). Labeled brains were imaged by confocal microscopy and maximum intensity projections are shown. Panels Q – R: Quantification of PSD95-immunoreactive post synaptic puncta in tissue volumes from (Panel Q) optic tectum and (Panel R) telencephalon. Data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.001***, 0.05*, 1-way ANOVA with Šidák multiple comparisons test. Figure 11 illustrates Brd4 contributes to microgliosis and synaptic pruning in transgenic zebrafish expressing human 0N / 4R-Tau and is expressed in human microglia. Panel A: Schematic depiction of Brd4 showing the positions of key functional domains in the human and zebrafish orthologues. Interspecies amino acid homology is indicated for each domain. The truncated products formed by a splice site-targeting morpholino (see Figure 34), and a stable germline 11-bp deletion in exon 4 of brd4 (Pt435) generated by CRISPR genome editing, are indicated. Panel B: Western blot showing expression of Brd4 (above) and ȕ-actin (loading control; below) in brains from WT zebrafish (lanes 1 Attorney Docket No.: 49045-0085WO1 / 05762 and 2) and brd4Pt435 / Pt435 zebrafish (lanes 3 and 4; abbreviated to brd4í / í in remaining panels). Panels C – D: Motor phenotypes in Ctrl, Tau, and Tau; brd4+ / ízebrafish. (Panel C) Example 1-minute swim vectors are shown above; graphs below show mean speed during the light phase of the VMR. Grey trace show mean frame-to- frame displacement scaled to show speed, colored markers and bars show 1-minute binned group mean ± SE, exactly as in Figure 8, panel B and Figure 9, panel F. (Panel D) Mean speed is shown for Ctrl, Tau, [Ctrl; brd4+ / í] and [Tau; brd4+ / í] zebrafish, quantified exactly as shown in Figure 8, panel E and Figure 10, panel A. Data points show individual zebrafish, bars show group mean ± SE. p<0.0001****, 0.05*, 1-way ANOVA with Šidák multiple comparisons test. Panel E: Acridine orange labeled spinal cord cells were quantified in Ctrl, Tau, [Ctrl; brd4+ / í] and [Tau; brd4+ / í] zebrafish as shown in Figure 6, panel E and Figure 10, panel K. Data points show individual zebrafish, bars show group mean ± SE. p<0.0001****, 1-way ANOVA with Šidák multiple comparisons test. Panel F: Microglia were quantified in Ctrl, [Ctrl; brd4+ / í],[Ctrl; brd4í / í], Tau, [Tau; brd4+ / í], and [Tau; brd4í / í] zebrafish as shown in Figure 6,panel J, Figure 10, panels L and M. Data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.01**, 1-way ANOVA with Šidák multiple comparisons test. Panels G – H: PSD95-immunoreactive synaptic puncta were quantified in Ctrl, Tau and [Tau; brd4+ / í] zebrafish in the optic tectum (Panel G) or telencephalon (Panel H) by whole mount immunofluorescence as shown in Figure 10, panels P, Q, and R. Data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 1-way ANOVA with Tukey multiple comparisons test. Panel I omitted. Panel J: Sections from control and PSP human substantia nigra were labeled for Brd4 (green), Iba1 (microglia; red) and nuclei (DAPI, blue) and imaged by confocal microscopy. Inset panels show Brd4- immunoreactive microglial nuclei from the main panels at higher magnification. Panel K: The proportion of Iba1-immunoreactive cells showing Brd4-immunoreactive nuclei was quantified in multiple sections from 4 control and 5 PSP cases in the substantia nigra and globus pallidus. Data points show proportion in individual cases, bars show group mean ± SE, comparison by unpaired 2-tailed t-test. The number of Brd4+ / Iba1+ microglia and total number of Iba1+ microglia analyzed in each group is indicated. Figure 12 illustrates transgene constructs and expression. Panel A: Schematic maps (not to scale) of the transgene constructs used to make Tau and Ctrl zebrafish. Key: UAS, upstream activating sequence; E1B, minimal promoter; 2A, self-cleaving viral 2A Attorney Docket No.: 49045-0085WO1 / 05762 peptide; 4R / 0N-hsMAPT, human cDNA encoding the Tau isoform lacking exons 2 and 3 but including exon 10; nls-mCherry, mCherry fluorescent protein fused to a nuclear localization signal; PolyA, polyadenylation signal. Positions of the open reading frames are indicated, illustrating the bicistronic cassette in the Tau + nls-mCherry construct. Panel B: Widefield epifluorescence image of a Tg(elavl3:gal4-vp16); Tg(UAS:hsa.MAPT-2a-nls-mCherry) ‘Tau’ zebrafish and its Tg(UAS:hsa.MAPT-2a-nls- mCherry) sibling at 5 dpf, illustrating robust transactivation of the transgene by the driver in the forebrain (FB), midbrain (MB), hindbrain (HB), retina (R), spinal cord (SC) and lateral line ganglion (LLG) by the panneuronal elavl3:gal4-vp16 driver. Figure 13 illustrates biological replicate survival cohorts for Tau zebrafish in comparison with controls. Figure 6, panel C (replicate #1) is reproduced alongside two further biological replicates (same alleles, different parents, different days), showing the survival of Tg(elavl3:gal4-vp16); Tg(UAS:hsa.MAPT-2a-nls-mCherry) zebrafish (Tau; red) in comparison with non-expressing siblings (see Table 4 for genotypes; Sib; black) and Tg(elavl3:gal4-vp16); Tg(UAS:2a-nls-mCherry) zebrafish (Ctrl; blue). The starting numbers of zebrafish in each cohort are indicated in each graph legend. These three cohorts provided the data for Figure 6, panel D. Figure 14 illustrates quantification of labeled CNS cells in zebrafish larval sections. Since larval zebrafish brains are small, exhaustive counts of labeled cells are possible in each section, but the area of brain shown varies according to the plane of each section, necessitating correction as follows: 1. Count immunolabeled cells within region of interest (brain) on each section (n=14 in image shown).2. Determine area of region of interest on each section (= 451703.6 ^m2= 0.452mm2in image shown).3. Calculate 7.4.C4 immunolabeled cells (microglia) per unit area: 14 x 106 / 451703.6 = 30.99 microglia / mm2for the section shown above.4. The same analysis is completed on 6 – 10 serial sections from each zebrafish: 5. Mean labeling density (cell / mm2) is calculated for each zebrafish. 6. Analysis is completed for 10 – 20 zebrafish in each experimental group at each time point to arrive at data shown in Figure 6, panel G and J.7. Identical analysis is carried out in immunofluorescence sections such as TUNEL labeling in Figure 6, panel F, except the area of interest is defined using anatomical landmarks in the DAPI labeled image, while blinded to the TUNEL image to increase rigor. The mask is then transferred to the TUNEL image for manual counting. Attorney Docket No.: 49045-0085WO1 / 05762 Figure 15 illustrates caspase 3 activation at 3 – 5 dpf in Tau zebrafish. Western blot of larval zebrafish lysates at 3 – 5 dpf were probed with an antibody to cleaved Caspae-3 (upper panel) and the blot then re-probed with an antibody to ȕ-Actin to confirm equal protein loading in all lanes (bottom panel). Lysates from Tau zebrafish at 3, 4 and 5 dpf, but not Ctrl zebrafish at the same ages, showed a prominent band at the expected size for cleaved (‘activated’) Caspase-3. A non-specific band running immediately below activated Caspase-3 is present in every lane as indicated. The 3dpf lanes from this blot are shown in the inset panel of Figure 6, panel G. Figure 16 illustrates human 4R-Tau truncation in Tau zebrafish. Western blots were made using the RIPA-soluble fraction from larval Tau and Ctrl zebrafish. Panel A: Tau zebrafish at 3 – 7 dpf were analyzed in comparison with Ctrl zebrafish at 5 dpf. The blot was probed with a monoclonal antibody to human Tau (Tau5) and a polyclonal antibody to ȕ-Actin to confirm equal protein loading in each lane (bottom panel). The full-length blot is shown for Tau. With increasing developmental age, the abundance of human 0N / 4R-Tau decreases steadily in the RIPA-soluble fraction, while the abundance of lower molecular weight truncation fragments of Tau increases steadily over time. (See Figure 7, panel G; the abundance of full-length human 0N / 4R-Tau does not change in the urea-soluble fraction over time, suggesting that Tau becomes progressively insoluble). Panel B: Tau and Ctrl zebrafish were analyzed at 7dpf using a monoclonal IgG1 antibody, TauC3, that specifically recognizes human Tau truncated by Caspase-3 at residue D421 and does not cross-react with full-length Tau. Faint bands (*) with sizes suggestive of 0N / 4R-Tau cleavage at D421 (possibly with other N-terminal cleavage events) are seen in Tau zebrafish. This correlates with the occasional TauC3- immonoreactive cells seen in histological sections (see Figure 7, panel H). Figure 17 illustrates biological replicate motor activity assays comparing Tau and Ctrl zebrafish.3 biological replicate experiments (same transgenic alleles, different parents, different days) comparing the mean speed of Tau and Ctrl zebrafish over 60 minutes of spontaneous swimming in bright white ambient illumination at 5 dpf. Data points show individual zebrafish, bars show group mean ± SE. groups and replicates were compared using 2-way ANOVA (experimental group, replicate) with Šidák multiple comparisons tests. The same data are shown three times for clarity. Panel A: There was a robust difference between Tau and Ctrl in each assay. Panel B: There was no difference between assays for Ctrl. Panel C: There was no difference between assays for Tau. Given Attorney Docket No.: 49045-0085WO1 / 05762 the absence of significant differences between the assays, it was admissible to combine these data to generate Figure 7, panels C, D, and E. Figure 18 illustrates additional movement parameters to accompany Figure 8, panels C, D, and E. Zebrafish motor activity at 5 dpf is discontinuous and stochastic. Consequently, mean speed (total scalar displacement / time) includes both movement events and rest periods. The large decrease in mean speed observed in Tau compared with Ctrl zebrafish (Figure 8, panel C) was accompanied by a substantial reduction in the proportion of each recording during which Tau zebrafish were moving (Figure 8, panel D), and a large increase in the mean interval between movement events in Tau zebrafish (Figure 8, panel E), which together accounted for most of the observed decrease in mean speed. However, less obvious changes were also apparent in the execution of movements. The graphs show two further parameters calculated from the same dataset as Figure 8, panels C – E. Panel A: Mean instantaneous speed during movement events. Panel B: Mean duration of movement events. In each graph, data points show individual zebrafish,bars show mean ± SE. ****p<10í15, ***p<0.001, Ctrl vs. Tau, 2-tailed unpaired t-testwith Welch’s correction for unequal variance. These data suggest that Tau zebrafish produced slower, shorter movement events than Ctrl. However, since the video recordings for these activity assays were captured at a low frame rate (4 frames / s), estimates of instantaneous speed and movement event duration are only approximations. A more rigorous evaluation of movement kinematics at high temporal resolution (1000 frames / s) is shown in Figure 7, panels G and H. Figure 19 illustrates three different control groups behave similarly in motor activity assays. In Figure 8, we show data comparing Tau and Ctrl zebrafish, which contain exactly the same complement of transgenes, with the exception that human Tau is not expressed in Ctrl zebrafish. However, since there is substantial genetic heterogeneity in WT zebrafish that can influence outcomes in neurological assays, the possibility that some of the phenotypes are caused by genetic variation unrelated to the transgene should be considered. To address this question, we tested two additional controls in this assay: (i) Siblings of Tau zebrafish that do not express the mCherry reporter (Sib; orange). These are a mixture of WT, driver and responder zebrafish that share background genetics with their Tau siblings. (ii) Tg(elavl3:gal4-vp16); Tg(UAS:egfp) zebrafish (GFP; green) that express a second responder allele unrelated to the Tg(UAS:2a-nls-mCherry) allele present in Ctrl zebrafish. Motor activity assays were run and analyzed identically to Attorney Docket No.: 49045-0085WO1 / 05762 those shown in Figure 8, panels C – E. The graphs show: Panel A: Mean speed. Panel B: Active swimming speed. Panel C: % time active. In each graph, data points show individual zebrafish, bars show mean ± SE. Comparison between groups was carried out using 1-way ANOVA with Tukey multiple comparison test. There were no statistically significant differences between the groups. Together with direct comparisons between Tau and each of these controls, and replicate phenotypes in multiple transgenic Tau lines, these data strongly support our interpretation that the observed phenotypes in Figure 8 are attributable to expression of human 0N / 4R-Tau. These data also support the use of these controls interchangeably in subsequent experiments. Figure 20 illustrates kinematic analysis of ‘O’-bend responses in Tau and Ctrl zebrafish. Ctrl and Tau zebrafish were analyzed at high temporal resolution using a 1000 frames / s video recording of their responses to abrupt transitions from bright white light ambient illumination to darkness. The characteristic high-angle turning movement that 5dpf zebrafish make in response to this stimulus is shown in Figure 8, panel G and is referred to as an ‘O’-bend as the zebrafish truncal outline resembles an upper-case letter O at peak curvature, when viewed from above. Videos were analyzed using a segmentation algorithm that identifies the zebrafish head, then projects three lines of equal length along its axis. The angles between the lines change when the zebrafish trunk curves during movement, allowing quantification of truncal curvature and angular velocity. In each graph, data points show individual zebrafish, bars show mean ± SE. ****p<10í15, Ctrl vs. Tau, 2-tailed unpaired t-test with Welch’s correction for unequal variance. Panel A: Response rate – for each zebrafish, the proportion of 40 stimuli to which a motor response was detected within 1s of the stimulus. Panel B: Response latency – the mean interval between stimulus and response for each zebrafish. Panel C: Response duration – the mean time period of contiguous frame transitions spanning each movement event. Panel D: Maximum curvature – peak truncal curvature for each response was determined and its mean was calculated for all responses for each zebrafish. This panel is also shown as Figure 8, panel H. Panel E: Peak angular velocity – the change in truncal curvature at video frame transitions was calculated and scaled to instantaneous angular velocity by dividing by the interval between frames. For each response, the peak angular trunk velocity was determined, and then the mean peak angular velocity for all responses was calculated for each zebrafish. Tau zebrafish showed a substantially reduced response rate and prolonged response latency to light- Attorney Docket No.: 49045-0085WO1 / 05762 dark transition, suggesting defective CNS processing of the stimulus. However, the kinematics (peak curvature and angular velocity) of the ‘O’-bend when executed were not different to controls, suggesting that – even though there are severe abnormalities of motor control – Tau zebrafish do not have overt neuromuscular paralysis. Figure 21 illustrates methodology and abnormalities of ocular movement in Tau zebrafish. Panel A: Cartoon illustrating the experimental configuration used to elicit optokinetic reflexes in Figure 8, panels J – N. Note that the visual stimulus is presented to the left (stimulated) eye only. Panel B: Comparison of ocular movement ranges for Ctrl and Tau zebrafish at 5dpf. The extremes of ocular movement for nasotemporal (left column) and temporonasal (right column) stimuli are shown. The long axis (dotted white line) and visual axis (solid white line) for each eye were calculated by the tracking algorithm and superimposed onto the image. The decreased angular range of movement in Tau zebrafish is reflected in the quantitative analysis shown in Figure 8, panel L. Panel C: Comparison of the stimulus angular velocity (green) and the left eye angular velocity for Ctrl (blue) and Tau (red) zebrafish. High-velocity saccadic positional resetting movements (whose angular velocity exceeds the axis limits) are indicated by ‘+’ and the Ctrl trace. In contrast to the Tau zebrafish, the slow phase movements of the Ctrl zebrafish closely mirror the stimulus. These data are reflected in the quantitative analysis of tracking gain shown in Figure 8, panel M. Panel D: Proportion of zebrafish lacking high-velocity saccadic movements in this assay. All Ctrl zebrafish showed some saccadic movements, which were absent from almost 60% of Tau zebrafish (***p<0.001, Fisher’s exact test). This analysis complements the quantitative analysis of saccade frequency shown in Figure 8, panel N. Panel E: Unilateral stimulus presentation in this assay allows analysis of the coordination between L and R eyes during the response. These graphs show how interocular gain (IOG; the ratio of change in angle of stimulated eye to change in angle of contralateral eye at each frame transition) and interocular concordance (IOC; the proportion of frame transitions at which there was movement of both eyes in the same direction) vary in Ctrl (blue) and Tau (red) zebrafish during the same segment of recording shown in panel C. Panels F and G: Quantitative analysis of mean IOG and mean IOC for Ctrl (blue) and Tau (red) zebrafish. In each graph, data points show individual zebrafish, bars show mean ± SE. ****p<0.0001, Ctrl vs. Tau, 2-tailed unpaired T-test with Welch’s correction for unequal variance. Attorney Docket No.: 49045-0085WO1 / 05762 Figure 22 illustrates abnormalities of the visual motor response in Tau zebrafish. In order to optimize an assay of motor activity for high-throughput screening, it was necessary to reduce variability as far as possible. This was achieved by using the zebrafish responses to alternating periods of bright ambient light and darkness, as evoked responses tend to be less variable than spontaneous behavior (as shown in Figure 8, panel B), and responses to multiple stimuli can be averaged. In addition, to simplify the breeding scheme for executing a screen, and further reduce variability by ensuring the controls shared similar genetic background and were exactly the same developmental age as the Tau zebrafish, the assay was optimized using non-expressing siblings as controls. This is valid because siblings showed identical responses to other controls in all other assays (Figure 19). Panel A: Motor responses for 31 Sib and 34 Tau zebrafish over three consecutive stimulus cycles of [10 minutes darkness + 10 minutes light] as shown above the graph. The gray traces show group centroid displacement at each video frame transition scaled to show mean speed (y-axis); the colored lines (Sib, black; Tau, red) show the same data averaged over a 50-frame moving window. Panel B: The three stimulus cycles were averaged to provide mean activity waveforms for each individual zebrafish over a single cycle; the averaged traces for each group are shown (gray). In each contiguous 1-minute time bin over the 20-minute averaged stimulus cycle, the mean speed for each zebrafish was calculated. This allowed calculation of group mean speed (solid markers) and SE (error bars) in each time bin across the averaged response cycle. These analyses substantially reduced response variability and increased statistical power to detect differences between smaller samples of zebrafish in each group, providing an essential basis for the screening assays shown in Figure 9. Since the difference between Ctrl and Tau was proportionately greater in the light phase of the response, and this was more quantitatively reproducible between biological replicates than the dark phase abnormalities, we used VMR light phase swimming speed as the readout for the screen. Figure 23 illustrates chemical modifier screen phase 1: Determination of maximum tolerated concentration for each chemical. Panel A: WT zebrafish embryos were collected after fertilization and housed in 6-well plates with daily changes of E3 buffer. Larvae were dechorionated at 2dpf and buffer replaced with fresh E3 containing chemical. The buffer was replaced with fresh E3 containing chemicals at 3dpf and 4dpf, then microscopy carried out at 5dpf to evaluate for viability (heartbeat) and morphology. Panel B: Each chemical was evaluated initially at 50^M concentration in the buffer. If Attorney Docket No.: 49045-0085WO1 / 05762 toxicity was found after exposure, the concentration was reduced. Iterative testing eventually provided a profile of maximum tolerated concentration (MTC) for each chemical in the library. Panel C: The bar graph shows the binned distribution (orange bars, left scale) and cumulative % distribution (blue line, right scale) of MTC for the epigenetic library. Figure 24 illustrates chemical modifier screen phase 2: Rescue of motor phenotype in Tau zebrafish. Panel A: Zebrafish embryos from a Tg(elavl3:gal4-vp16) x Tg(UAS:hsa.MAPT-2a-nls-mCherry) cross were collected and housed in 6-well plates with daily changes of E3 buffer. Embryos were genotyped by mCherry fluorescence at 1dpf and sorted into Tau and Sibling groups. The larvae were then dechorionated at 2dpf, genotypes verified, and buffer replaced with fresh E3 containing chemical. There were 7 groups of Tau zebrafish: 6 of the groups received E3 containing a different library chemical at its MTC defined in phase 1 of the screen, the 7th group received E3 only. A single group of 12 Sib zebrafish also received E3. Buffer was replaced with fresh E3 ± chemical at 3dpf and 4dpf. Panel B: At 5dpf, chemical was washed off and replaced with fresh E3 and the VMR elicited as shown in Figure 22 for quantitative analysis as shown in Figure 25. The entire library was tested, 6 compounds at a time, in this way. Figure 25 illustrates algorithm logic for analysis of video tracking data from chemical rescue screening assays. The flowchart depicts the algorithm used to calculate rescue of the abnormal VMR phenotype in Tau zebrafish by chemical exposure. Calculations are split into 4 consecutive processes: 1. Movement calculations (blue) – individual zebrafish responses are averaged over 3 cycles of dark-light stimuli as shown in Figure 22, then mean speed for each zebrafish calculated in contiguous 30-second time bins across the averaged stimulus cycle. Mean and SE speed are then calculated for each experimental group in each time bin across the stimulus cycle. 2. Assay quality control (QC) calculations (yellow) – the responses of untreated Tau and Sib zebrafish are compared to ensure that the phenotype in Tau zebrafish was sufficiently robust to make rescue calculations meaningful. Individual time bins where SpeedSib í SpeedTau < 0.3 mm / s are assigned QC = 0 and rejected. Each accepted time bin is then analyzed to ensure that SpeedSib and SpeedTau are statistically significantly different at p<0.01 by t- test; if so, the time bin is assigned a QC score of 1 and accepted. If p > 0.05, the bin is assigned QC = 0 and rejected. For 0.01 < p < 0.05, QC is weighted linearly between 1 and 0. Mean QC is then calculated for the assay. If QC < 0.95 the assay has failed quality Attorney Docket No.: 49045-0085WO1 / 05762 control and no further analysis is possible. This stringent QC process overall rejected < 5% assays, and ensured that data pertaining to phenotypic rescue were only accepted as part of the screen if the model behaved exactly as expected.3. Rescue calculations (orange) – this calculates % rescue for each chemical in each time bin according to the formula shown in Figure 9, panel C multiplied by the QC value for the bin. For each chemical and each time bin, if SpeedTau and SpeedTau+Chemical are significantly different at p < 0.05 by t-test, the value for % rescue in that time bin is accepted. If p > 0.1, rescue is set to 0 for that time bin. For 0.1 < p < 0.05, rescue is weighted by (t í 1)3. These stringent criteria for accepting data points minimized any contribution to overall rescue score from time bins in which effects were marginal. Mean rescue for each chemical is then calculated for the light and dark phases of the VMR, omitting any time bins that were rejected at stage 2 (QC). 4. Outputs (green) – QC metrics and binned rescue data are output graphically (see Figure 26) and numerical data are written into a spreadsheet for downstream analysis. Figure 26 illustrates example outputs from one screening assay. Panel A: Mean ± SE speed of 12 Tau (red) and 12 Sib (blue) zebrafish in 30-second time bins, averaged across 3 x 20-minute dark-light stimulus cycles (first graph). There is a robust phenotype but overlap between Tau and Sib at the start of the dark phase would make rescue calculations impossible in these time bins. This is reflected in the QC score (second graph), which rejects bins 2 – 4 of the dark phase responses. The assay window (difference between Sib and Tau; third graph) and phenotype ratio (assay window as a fraction of Sib response) show the effect size attributable to Tau across the stimulus cycle. Overall, the VMR light phase in this experimental run performed well as an assay to detect chemical rescue of neurological function. Panel B: Automated outputs showing the effect of each of 6 chemicals from the assay shown in panel A. In the top row, the responses of Tau zebrafish exposed to chemical (red) are superimposed on the Tau (blue) and Sib (green) controls. None of the chemicals improved the motor function of Tau zebrafish. The second and third rows show calculations for phenotypic rescue, either without (middle row) or with (bottom row) removal of points failing QC and correction for whether the effect was significant in each time bin. Compounds marked * were subsequently retested at lower concentrations. Figure 27 illustrates hits recovered from epigenetic library screen. The chemical structures, maximum tolerated concentrations, phenotypic rescue, and molecular targets Attorney Docket No.: 49045-0085WO1 / 05762 are summarized for compounds if interest identified in this screen. Panel A: The threecompounds that satisfied a priori criteria for a ‘hit.’ Panel B: (í)JQ1 is a negative controlstereoisomer of (+)JQ1; its lack of activity in this assay is strongly suggestive that (+)JQ1 exerts its effect through a specific receptor-ligand interaction. Panel C: Other compounds that did not satisfy a priori criteria as ‘hits’ but appeared to show some activity in this assay and may therefore be of interest. Figure 28 illustrates ranked phenotypic rescue activity by target. Rescue data grouped by pharmacological target, similar to Figure 9, panel E and Figure 19, but analyzed by rank rather than % rescue. Panel A: Table showing mean % rescue and median rank by pharmacological target. Panel B: Graph showing rescue rank by target. Data points show individual chemicals, bars show median and quartiles. The data show similar trends to Figure 9, panel E, with BET bromodomain inhibitors showing the best median rescue rank. Figure 29 illustrates concentration-dependent rescue of motor function in Tau zebrafish by (+)JQ1. Similar analyses to Figure 8, panels C – E and Figure 18, showing how abnormalities of motor activity in Tau zebrafish at 5dpf were altered by exposure to (+)JQ1 between 2 – 5 dpf, at incremental concentrations from 0.25 to 1.5^M. In each graph, data points show individual zebrafish, bars show mean ± SE. ****p<0.0001, Ctrl vs. experimental group, 1-way ANOVA with Dunnett’s multiple comparison test. Panel A: Mean speed (scalar distance traveled by zebrafish centroid / time of assay). Panel B: Active swimming speed (mean scalar speed of zebrafish centroid during movement events). Panel C: Time active % (proportion of video frame transitions at which displacement occurred). Panel D: Mean active duration (mean duration of movement events). Panel E: Mean inter-movement interval (mean duration of stationary periods between movements). In each case, motor abnormalities in Tau zebrafish returned progressively towards control values with increasing concentrations of (+)JQ1. Figure 30 illustrates (+)JQ1 improves survival in three independent cohorts of Tau zebrafish. The [Tau] and [Tau + (+)JQ1] groups are reproduced from 5G (replicate #1), alongside two further biological replicates (same alleles, different parents, different days; each cohort shown here corresponds to the same cohort in Figure 31, but the Tau and Ctrl zebrafish are separated into different figures for clarity). These three replicate cohorts show the survival of Tau zebrafish (Tau; red) in comparison with Tau zebrafish exposed to 1^M (+)JQ1 (Tau + JQ1; green). The starting numbers of zebrafish in each Attorney Docket No.: 49045-0085WO1 / 05762 cohort are indicated in each graph legend. These three cohorts provided data for Figure 10, panels G, H, and J. ****p<0.00001 Tau vs. Tau + JQ1, Mantel-Cox test. Figure 31 illustrates (+)JQ1 does not alter survival in three independent cohorts of Ctrl zebrafish. The [Ctrl] and [Ctrl + (+)JQ1] groups are reproduced from 5G (replicate #1), alongside two further biological replicates (the three cohorts shown here correspond to the same cohorts as Figure 30, but the Tau and Ctrl groups are separated into different figures for clarity). These three individual cohorts show that survival of untreated (blue) and (+)JQ1-exposed (1^M; gold) Ctrl zebrafish was similar. The starting numbers of zebrafish in each cohort are indicated in each graph legend. These three cohorts provided data for Figure 10, panels G and H. Figure 32 illustrates volumetric imaging of synapse abundance in zebrafish larval brain. PSD95 whole mount immunolabeling and volumetric image acquisition in the telencephalon (upper row) and optic tectum (lower row) were carried out in Ctrl (left), Tau (center) and [Tau + (+)JQ1] (right) zebrafish. The figure shows the same samples as Figure 10, panel P, but the images here are pseudocolored to show depth within the image stack, illustrating the large volume of PSD95 expression data that was captured by this method. Figure 33 illustrates (+)JQ1 does not rescue OKR deficits in Tau zebrafish. Similar analyses to Figure 8 panels J – N and Figure 21, showing quantification of optokinetic reflexes in Ctrl and Tau zebrafish at 5dpf after no treatment, or exposure to (+)JQ1 or (í)JQ1 from 2 – 5 dpf. In Panels A – E, data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.01**, 1-way ANOVA with Šidák multiple comparison test. In panel F, bars show proportion ± SE(p) of zebrafish with and without saccadic movements during exposure to OKR stimuli. In contrast to motor function and survival, there was no evidence that (+)JQ1 rescued OKR deficits in the Tau model after treatment at these time points. Figure 34 illustrates transient knockdown of Brd4 in Tau zebrafish mitigates microgliosis at 4dpf. Morpholino oligonucleotides (MO) targeting brd4 expression were used to evaluate the role of Brd4 in microgliosis in Tau zebrafish. Two different MO were used to increase confidence that any observed effects were attributable to loss of Brd4 expression. Panel A: MO targeting the translational start site and adjacent 5’UTR of the zebrafish brd4 mRNA. This is predicted to decrease translation of Brd4. Panel B: MO targeting the splice acceptor sequence at the boundary between intron 5 and exon 6 of the Attorney Docket No.: 49045-0085WO1 / 05762 primary brd4 transcript. This is predicted to skip exon 6 during splicing and introduce a frame shift mutation and premature stop codon into the mRNA, as the splice boundary phases of i5 / e6 and i6 / e7 differ with respect to the ORF. MO were microinjected at the single-cell stage and both MO were compared with a non-targeting negative control MO (neg MO) to exclude effects caused by microinjection, injection buffer, or nonspecific effects attributable to MO exposure. In the upper panels, western blots were made from lysates of 2 dpf control, brd4 MO-microinjected and neg MO-microinjected zebrafish, and then probed with an antibody to zebrafish Brd4 (obtained from Dr. Igor Dawid, NIH). At this developmental point, the antibody recognizes a protein of 160kDa corresponding to the expected size of Brd4, in addition to other smaller proteins whose identity is unclear (#). In each case, the 160kDa band disappeared after brd4 MO microinjection but not neg MO injection, confirming that Brd4 was targeted by both MO. In the lower panels, 7.4.C4-immunoreactive microglia in sections were quantified by immunohistochemistry at 4dpf, identically to Figure 6, panel J, in Ctrl or Tau zebrafish that were untreated, or microinjected with either Brd4 or non-targeting MO. In each graph, data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.001***, 0.05*, 1- way ANOVA with Šidák multiple comparison test. The large increase in microglial abundance in Tau zebrafish was prevented by Brd4 knockdown, suggesting Brd4 is the target through which (+)JQ1 prevents microgliosis in this model. Figure 35 illustrates generation of a stable brd4 null allele using CRISPR genome editing. Panel A: The brd4Pt435 allele (SEQ ID NO:1) described in the paper was made using CRISPR / Cas9. A gRNA sequence (SEQ ID NO:2) was identified to target a PAM site within a BamHI restriction enzyme site in exon 6 of the brd4 gene. This was chosen to allow easy genotyping by showing loss of the BamHI site in a PCR amplicon spanning the mutation, without necessitating DNA sequencing. Panel B: Multiple mutant alleles were recovered at the F1 stage, including small deletions and insertions as shown. Sequences shown include Brd4 WT (SEQ ID NO:1), a Brd4 having a 8bp deletion (SEQ ID NO:3), a Brd4 having a 11bp deletion (SEQ ID NO:4), a Brd4 having a 27bp deletion (SEQ ID NO:5), a Brd4 having a 5bp deletion and a 5bp insertion (SEQ ID NO:6), a Brd4 having a 5bp insertions (SEQ ID NO:7), and a Brd4 having a 45bp insertion (SEQ ID NO:8). The 11bp deletion was chosen for further studies, as it abolished Brd4 expression (Figure 11, panel B). Panel C: The Pt43511bp deletion allele causes a frameshift mutation leading to a premature stop codon within exon 6 (SEQ ID NO:9; Attorney Docket No.: 49045-0085WO1 / 05762 with the encoded amino acid (SEQ ID NO:10)). The Brd4 WT sequence (SEQ ID NO:11; with the encoded amino acid (SEQ ID NO:12)) is shown for reference. Panel D: The predicted translation of the Pt435 allele leads to a severely truncated protein that ends after the first bromodomain of Brd4. Genetic studies suggest this is a null allele (see below) and not a dominant negative, as there is no phenotype in heterozygotes. The protein fragment may be unstable. Figure 36 illustrates developmental phenotypes in brd4í / í zebrafish. Panel A: Genotypes from three independent clutches (same allele, different parents) of brd4í / í zebrafish at 5dpf. There was no significant difference between the observed genotypes and the expected Mendelian ratios. Panel B: Genotype proportions for the three clutches overall did not differ from the expected Mendelian ratios at 5dpf. However, no homozygous brd4í / í zebrafish adults were identified from >100 offspring of brd4+ / í in- crosses that were genotyped, strongly suggesting that brd4í / í zebrafish are not viable to adulthood. Panel C: Morphological changes in brd4í / í zebrafish, including jaw region dysgenesis, pericardial edema, failure of swim bladder insufflation, and kyphotic spinal curvature. Panel D: Relationship between genotype and presence of a swim bladder. There was a strong statistically-significant association between brd4í / í genotype and failure of swim bladder insufflation in each of the three clutches analyzed in panel A. Panel E: Genotype proportions for zebrafish from each of the three clutches without an observable swim bladder at 5dpf. The brd4í / í genotype was strongly associated with absent swim bladder. Figure 37 illustrates motor activity abnormalities in brd4í / í zebrafish. 5dpf larvae resulting from brd4+ / í in-crosses were analyzed in a 96-well plate motor activity assay similar to Figure 8, panels A – F, and then each larva was genotyped by PCR amplification and BamHI restriction digest for the brd4Pt435 allele. In each graph, data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.01**, 1-way ANOVA with Dunnett multiple comparison test. Panel A: Mean speed (scalar distance traveled by zebrafish centroid / time of assay). Panel B: Active swimming speed (mean scalar speed of zebrafish centroid during movement events). Panel C: Time active % (proportion of video frame transitions at which centroid displacement occurred). Panel D: Mean active duration (mean duration of movement events). Panel E: Mean inter- movement interval (mean duration of stationary periods between movements). Together with Figure 36, these data suggest that analysis of brd4 as a genetic modifier of the Tau Attorney Docket No.: 49045-0085WO1 / 05762 phenotype would be difficult to interpret using homozygous brd4í / í mutants that have morphological and motor phenotypes of their own. However, heterozygous brd4+ / ímutants that have no detectable abnormalities should provide a reasonable way to determine if decreased Brd4 expression modulates the phenotype in Tau zebrafish. Figure 38 illustrates heterozygous brd4+ / í mutations partially rescue motor phenotypes in Tau zebrafish.5dpf Tau and Ctrl larvae resulting from [Tg(elavl3:gal4- vp16); brd4+ / Pt435] x [Tg(UAS:hsa.MAPT-2a-nls-mCherry) or Tg(UAS: 2a-nls- mCherry)] crosses were analyzed in a 96-well plate motor activity assay similar to Figure 8, panels A – F, and then each larva was genotyped by PCR amplification and BamHI restriction digest for the brd4Pt435 allele. In each graph, data points show individual zebrafish, bars show mean ± SE. p<0.0001****, 0.01**, 1-way ANOVA multiple comparison test. Panel A: Mean speed (scalar distance traveled by zebrafish centroid / time of assay). Panel B: Active swimming speed (mean scalar speed of zebrafish centroid during movement events). Panel C: Time active % (proportion of video frame transitions at which centroid displacement occurred). Panel D: Mean active duration (mean duration of movement events). Panel E: Mean inter-movement interval (mean duration of stationary periods between movements). These data show that heterozygous brd4+ / í mutations partially rescue mean speed, % time active and inter-movement interval in Tau zebrafish. The less prominent abnormalities in active swimming speed and mean active duration found in Tau zebrafish in Figure 18 were not apparent in these clutches. Figure 39 illustrates heterozygous brd4+ / í mutation does not recue OKR deficits in Tau zebrafish. Tg(UAS: 2a-nls-mCherry)] crosses were analyzed in OKR assays similar to Figure 8 panels J – N and Figures 21 and 33, and then each larva was genotyped by PCR amplification and BamHI restriction digest for the brd4Pt435 allele. In panels A – E, data points show zebrafish, bars show mean ± SE. p<0.0001****, 0.01**, 1-way ANOVA with Šidák multiple comparison test. In panel F, bars show proportion ± SE(p) of zebrafish with and without saccadic movements during exposure to OKR stimuli. Similar to findings with (+)JQ1 exposure, decreased Brd4 expression did not rescue OKR deficits in the Tau model at the time points evaluated. Figure 40 illustrates rescue of synaptic puncta in Tau zebrafish with heterozygous brd4+ / í mutations.5dpf Tau and Ctrl larvae resulting from [Tg(elavl3:gal4-vp16); brd4+ / Pt435] x [Tg(UAS:hsa.MAPT-2a-nls-mCherry)] or [Tg(elavl3:gal4-vp16) x Attorney Docket No.: 49045-0085WO1 / 05762 Tg(UAS: 2a-nls-mCherry)] crosses were genotyped by PCR amplification and BamHI restriction digest for the brd4Pt435 allele, and then fixed for PSD95 immunofluorescence. These example images are pseudocolored to show depth within the image stack, and accompany the quantitative analysis shown in Figure 11, panels G and H. Figure 41 illustrates Brd4 expression in the nuclei of human substantia nigra microglia. Individual fluorescence channels for the images shown in Figure 11, panel J are separated and shown in monochrome for clarity. Image fields are shown from a control brain (top row) and a PSP brain (bottom row). Individual DAPI labeled nuclei (column 1) that were immunoreactive for Brd4 (column 2) and were located within Iba1- immunoreactive microglia (column 3) are indicated with white arrows in each image of a set. The overlaid pseudocolored images are shown in column 4. Figure 42 illustrates Brd4 expression in microglia from multiple control and PSP cases. Human control and PSP brain sections from two brain regions characteristically affected by PSP pathology – the substantia nigra and globus pallidus – were immunolabeled for Brd4 (green), Iba1 (microglial marker; red) and counterlabeled with DAPI (nuclei; blue). Images were acquired by confocal microcopy. Panel A: Examples of Brd4+, Iba1+ microglia are shown from the substantia nigra of each of 4 control brains. Panel B: Examples of Brd4+, Iba1+ microglia are shown from the substantia nigra of each of 5 PSP brains. Panel C: There was no significant overall increase in the density of Iba1-labeled microglia in either the substantia nigra or the globus pallidus of PSP brains in this small sample, despite the characteristic morphological differences (retracted processes, prominent perinuclear cytoplasm) suggestive of activation seen in the images from PSP brains. DETAILED DESCRIPTION It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described representative embodiments. Thus, the following more detailed description of the representative embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely illustrative of representative embodiments. Reference throughout this specification to “one embodiment” or “an Attorney Docket No.: 49045-0085WO1 / 05762 embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such compounds and equivalents thereof known to those skilled in the art, and so forth, and reference to “the compound” is a reference to one or more such compounds and equivalents thereof known to those skilled in the art, and so forth. 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, each separate value, as well as intermediate ranges, are incorporated into the specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text. In a number of embodiments, a new molecular target is identified via a phenotype-based chemical modifier screen in a zebrafish model. The target may, for example, mediate microglial activation in tauopathy and works through a previously unexplored mechanism. Moreover, a number of compounds are identified for treatment of tauopathies, with various molecular targets. A novel approach to identifying compounds for treatments uses software for tracking the movements of zebrafish in, for example, 96-well plates coupled with analytical software to enable truly phenotype- driven discovery in chemical screens. The compositions, systems, and methods hereof may be transformative in the field. To develop a zebrafish progressive supranuclear palsy or PSP model well suited Attorney Docket No.: 49045-0085WO1 / 05762 for chemical screening applications, Gal4-UAS genetics can be employed. The development of the transgenic zebrafish model hereof and the use there in representative screening studies is described and can be performed as set forth in Example 1. A UAS responder cassette encoded WT human 0N / 4R-Tau, together with, for example, nls- mCherry can be used to allow rapid identification of transgenic zebrafish by fluorescence microscopy in downstream applications. Use of a 2A peptide ensured that human 0N / 4R- Tau and nls-mCherry were expressed as separate proteins (directed to different subcellular compartments) from the same mRNA transcript, thereby avoiding the potential for a fluorescent fusion protein to alter the pathophysiological properties of Tau. By generating Tg(UAS:hsa.MAPT-p2A-nls-mCherry) lines in the absence of Gal4- induced expression, stable heritable transgene alleles were isolated that could be propagated readily. Lines were then selected that showed robust transactivation when crossed with a pan-neuronal Gal4 driver to yield Tg(elavl3:Gal4-VP16); Tg(UAS:hsa.MAPT-p2A-nls-mCherry) ‘Tau’ zebrafish. Tg(elavl3:Gal4-VP16); Tg(UAS:p2A-nls-mCherry) ‘Ctrl’ zebrafish were generated to provide controls with the same complement of transgenes as Tau zebrafish but lacking human 0N / 4R-Tau. Transgenic zebrafish were similarly developed expressing human P301L 0N / 4R-Tau. Crossing Tg(elavl3:gal4-vp16) driver and Tg(UAS:hsa.MAPT-p2A-nls-mCherry) responder lines (each alone healthy) together yielded double transgenic “Tau” zebrafish, in which the UAS enhancer is trans-activated in neurons by Gal4-VP16 expressed from the elavl3 driver, resulting in Tau expression. The mCherry (or other imaging / fluorescent) reporter allowed non-invasive identification of double transgenic human Tau-expressing zebrafish. As described herein, in the transgenic zebrafish hereof, mCherry with an associated targeting signal is separated from Tau post-translationally by a self-cleaving peptide such as a viral 2A peptide, so the two resulting proteins (one of which is a fluorescent protein such as mCherry) are expressed independently in separate tissue compartments. In a number of embodiments, nls-mCherry was used after the 2A peptide so the reporter was expressed in the nucleus. The lifespan of Tau zebrafish (median survival 8-9 days in replicate cohorts) was severely attenuated in comparison with Ctrl zebrafish or non-expressing siblings (both control lines survive to aged adulthood, >2 years). Three complementary assays – failure of acridine orange exclusion in vivo, TUNEL labeling of apoptotic cells in histological sections, and detection of Capsase-3 cleavage – demonstrated a robust increase in Attorney Docket No.: 49045-0085WO1 / 05762 neuronal death in Tau compared with Ctrl zebrafish between 2-7 days post-fertilization (see Example 1). Peak cell death was detected in Tau zebrafish in all three assays at 3-4 dpf, with up to 10-fold excess apoptotic cells compared with Ctrl. By 5 dpf, decreases in the expression of dopaminergic (Tyrosine Hydroxylase; TH) and GABAergic (Glutamic Acid Decarboxylase; GAD) neuronal markers, and in both pre- (Synaptophysin; SYP) and post- (Post-Synaptic Density 95 kDa; PSD95) synaptic markers, were apparent in Tau zebrafish compared with Ctrl by western blot. Immunohistochemistry also showed a large (> 2-fold) increase in brain microglia in Tau zebrafish compared with Ctrl between 3-6 dpf. Together, these data show that conditional expression of human 0N / 4R-Tau in transgenic zebrafish causes impaired survival, neurodegeneration, loss of neurochemical and synaptic markers, and neuroinflammation, which are key features of PSP. Automated machine vision measurement of zebrafish motor activity in 96-well plates at 5 dpf showed that Tau zebrafish were profoundly hypokinetic in comparison with Ctrl zebrafish during spontaneous movement in bright ambient illumination over prolonged recordings. Procedures for such automated measurements are described elsewhere (Zhou, Y., Cattley, R.T., Cario, C.L., Bai, Q. & Burton, E.A. Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications. Nat Protoc 9, 1533-48 (2014); Farrell, T.C. et al. Evaluation of spontaneous propulsive movement as a screening tool to detect rescue of Parkinsonism phenotypes in zebrafish models. Neurobiol Dis 44, 9-18 (2011); and Cario, C.L., Farrell, T.C., Milanese, C. & Burton, E.A. Automated measurement of zebrafish larval movement. J Physiol 589, 3703-8 (2011)), included as Appendices 2, 3 and 4, respectively, in U.S. Provisional Patent Application 63 / 518,592, all three of which are incorporated herein by reference in their entireties. Mean scalar speed (centroid displacement / time) is an integrative measurement of zebrafish motility, reflecting both the speed and size of individual movements and the frequency with which they are executed. A robust decrease in mean speed in Tau zebrafish compared with Ctrl zebrafish was observed in multiple replicate assays and was attributable to: (i) a large increase in the time interval between movements, combined with a small decrease in the duration of movement events, resulting in a decreased proportion of the assay during which Tau zebrafish were motile; and (ii) a modest decrease in the speed of individual movements by Tau zebrafish. High temporal and spatial resolution kinematic analysis of motor responses evoked by abrupt ambient light-dark transition (see, Hossainian, D., et al., Quantification Attorney Docket No.: 49045-0085WO1 / 05762 of functional recovery in a larval zebrafish model of spinal cord injury, Neuroscience Research, (11):2044-2054 (2022), which is (a) included as Appendix 5 in U.S. Provisional Patent Application 63 / 518,592 and (b) incorporated herein by reference in its entirety) showed that peak truncal curvature and peak truncal angular velocity were unchanged in Tau zebrafish compared with Ctrl during high-angle ‘O’-bend turning movements, even though the response rate to illumination transitions was lower and the latency to response prolonged. The data suggest that hypokinesia in Tau zebrafish is not attributable to neuromuscular paralysis, but is caused by reduced movement initiations, with possible parallels to the akinetic motor disorder seen in PSP patients. In view of the prominent oculomotor disorder for which PSP is named, the eye movements of Tau zebrafish were next evaluated by analyzing optokinetic reflexes. Procedures for such automated measurements are described elsewhere (Shao, E., Scheetz, S.D., Xie, W. & Burton, E.A. Modulation of the zebrafish optokinetic reflex by pharmacologic agents targeting GABAA receptors. Neurosci Lett 671, 33-37 (2018); and Scheetz, S.D. et al. An open-source method to analyze optokinetic reflex responses in larval zebrafish. J Neurosci Methods 293, 329-337 (2018), included as Appendices 6 and 7, respectively, in U.S. Provisional Patent Application 63 / 518,592, both of which are incorporated herein by reference in their entireties). Projection of an animated grating pattern on a screen filling the zebrafish visual field provokes characteristic cycles of slow ocular movements in the stimulus direction (that serve to stabilize the moving image in the retina) and rapid positional resetting movements (saccades) in the opposite direction. Compared with Ctrl zebrafish, Tau zebrafish showed a decreased range of ocular movement, reflex tracking gain, and saccade frequency, in addition to disrupted coordination between movements of the two eyes. Complete loss of saccades during OKR was observed in 60% Tau zebrafish, resembling a characteristic clinical abnormality found in PSP patients. Overall, the data show that Tau zebrafish develop robust OKR deficits reminiscent of those found in PSP. Studies were conducted to determine whether automated measurement of hypokinesia could be used as an unbiased and quantitative screening endpoint to detect interventions that rescue the neurological phenotype of Tau zebrafish. For these analyses, Tau zebrafish were compared with non-expressing siblings, to simplify breeding for subsequent screening. This approach is valid, as Ctrl zebrafish and non- expressing siblings of Tau zebrafish showed identical responses in 96-well plate motor Attorney Docket No.: 49045-0085WO1 / 05762 assays. Variability in measured motor activity was minimized by averaging the responses to multiple cycles of alternating ambient illumination and darkness. Mean light phase swimming speed showed the largest and most replicable difference between Tau zebrafish and their siblings in these assays, but analysis of single zebrafish using this metric as an endpoint did not provide adequate performance as a screening assay. Averaging the responses of multiple zebrafish in each treatment group substantially improved assay performance at the expense of throughput. An optimized group size of 12 Tau zebrafish allowed 6 compounds to be tested in parallel with controls in each 96- well plate, with a consistently positive Z’ indicating a remarkable level of performance for a neurobehavioral assay. A library of 147 inhibitors of epigenetic readers, writers, and erasers was selected, because the compounds of the library target cellular physiology that is relatively unexplored in tauopathy, and the modest library size is manageable for an initial screen against the Tau model. Screening was completed in two phases: (i) the maximum tolerated concentration (MTC) of each chemical was defined experimentally in WT zebrafish using survival and morphological endpoints; and (ii) each chemical was then tested at MTC in 12 Tau zebrafish, and their mean light phase swimming speed normalized to the phenotypic window defined by 12 untreated Tau zebrafish and 12 non- expressing siblings in each assay. The controls provided stringent inbuilt assay QC, and normalization of chemical responses to within-assay controls allowed comparison between assays. Both QC benchmarks and data analysis were automated to eliminate bias and accelerate workflow, and chemicals were screened with their identities masked until the entire library had been analyzed. Mean ± SE % phenotypic rescue for each compound is shown in Figure 1 in comparison with untreated Tau zebrafish (0% rescue) and non-expressing siblings (100% rescue). Three of the compounds tested satisfied an a priori definition of a ‘hit’ (mean library rescue + 3 SD; Figure 1 and Table 1 inset), whereas the remainder were distributed around the untreated Tau group. The highest performing hit in Figure 1, (+)JQ1 (Compound #1), is known to disrupt binding of acetylated lysine residues to the bromodomains of bromo- and extraterminal-domain containing (BET) proteins, which regulate transcriptional responses to histone acetylation. The other two hits were Trichostatin-A (Compound #2; which is known to inhibit multiple histone deacetylases, HDACs) and 2,4-DPD (Compound #3; which is known to inhibit hypoxia-inducible factor-1Į prolyl hydroxylase). Clustering Attorney Docket No.: 49045-0085WO1 / 05762 the compounds by pharmacological target showed that BET, multi-HDAC, or DNA methyltransferase inhibitors overall improved motor function significantly. There was substantial variability between compounds within each target class that may reflect variation in their pharmacokinetics or differential binding affinity for zebrafish proteins. Repurchased (+)JQ1 rescued Tau motor function in a replicable, concentration-dependent manner, formally confirming its identity. The inactive stereoisomer (í)JQ1 did not rescue motor function in Tau zebrafish demonstrating the requirement of a specific ligand-bromodomain interaction for activity. (+)JQ1 did not increase the swimming speed of Ctrl zebrafish, excluding non-specific stimulation of zebrafish motility as the explanation for the observations. Exposure to (+)JQ1 did not reduce 0N / 4R-Tau levels, alter Tau phosphorylation (determined by electrophoretic mobility or immunoreactivity to phosphoepitope-specific antibodies), or silence transgene expression. Together the data suggest that (+)JQ1 acts to inhibit pathophysiology in this model conceptually downstream of Tau. Median survival of Tau zebrafish was increased 15-20% by exposure to (+)JQ1, which did not alter survival of Ctrl zebrafish. (+)JQ1 did not decrease acridine orange labeled cells, indicating that hypokinesia is not caused by spinal cord cell death in this model. However, (+)JQ1, but not (í)JQ1, decreased CNS microglial abundance substantially in Tau zebrafish at 5 dpf. Without limitation to any mechanism, the reduced density of microglia in (+)JQ1-exposed Tau zebrafish may delay phagocytic removal of apoptotic neurons, potentially accounting for the modest increase in AO-labeled cells observed. Since (+)JQ1 prevents microgliosis in Tau zebrafish, it was further studied whether improvement of motor function might be explained by (+)JQ1 rescuing microglial synapse elimination. Microglial synaptic pruning was identified recently as an important mechanism in neurodegenerative disease models and synaptic loss was previously demonstrated histopathologically in PSP. Western blot analysis showed that exposure to (+)JQ1 restored expression of the post-synaptic marker PSD95 in Tau zebrafish. Volumetric imaging in whole mount brains, coupled with unbiased automated image analysis, showed that the density of PSD-95 immunoreactive synaptic puncta in Tau zebrafish was decreased in both the optic tectum and dorsal telencephalon compared with Ctrl. This deficit in synaptic abundance was rescued by exposure to (+)JQ1. Those data together suggest that (+)JQ1 prevents microglial elimination of synapses in Tau zebrafish, possibly accounting for its effect in improving motor function and survival. Attorney Docket No.: 49045-0085WO1 / 05762 Several components of the Tau zebrafish phenotype, including decreased motor activity in the dark phase of the VMR and disrupted OKRs in Tau zebrafish, were not rescued by (+)JQ1. Such phenotypes may be mediated by a different mechanism. The molecular target of (+)JQ1 in the zebrafish Tau model was verified using reverse genetics. Previous work showed that (+)JQ1 binds with high affinity to the BET protein Brd4, which has been implicated in inflammatory signaling in other contexts. Zebrafish have a single highly-conserved orthologue of human Brd4. Antisense morpholino oligonucleotides (MO) that target splicing or translation of the zebrafish brd4 mRNA decreased Brd4 expression and mitigated microgliosis in Tau zebrafish at 3-4 dpf. However, progressive loss of MO during cell division following microinjection means that gene knockdown only lasts until 2-3 dpf. To test the role of Brd4 in Tau zebrafish at later timepoints when phenotypic abnormalities are most prominent, a stable 11-bp deletion in exon 4 of brd4 was engineered that abrogated Brd4 expression (allele designation Pt435), using Cas9 / CRISPR. Homozygous brd4í / ímutants lacking Brd4 showed morphological and motor deficits, and were not viable. However, heterozygous brd4+ / ízebrafish showed no overt abnormalities, and [Ctrl; brd4+ / í] zebrafish showed motor function to their [Ctrl; brd4+ / +] siblings, allowing testing of the effect of the heterozygous brd4 mutation on the neurological phenotypes of Tau zebrafish. The VMR light phase mean speed of [Tau; brd4+ / í] zebrafish was significantly higher than [Tau; brd4+ / +] siblings, demonstrating that decreased Brd4 expression partially rescued hypokinesia in Tau zebrafish. Similar to (+)JQ1 exposure, reduced Brd4 expression did not rescue acridine orange labeled spinal cord cells or OKR deficits in Tau zebrafish. However, there was a robust decrease in CNS microglial abundance in zebrafish harboring brd4 mutations. The density of brain microglia in Tau zebrafish was highest in brd4+ / +zebrafish, decreased in brd4+ / íheterozygotes, and lowest in homozygous brd4í / ímutants. The data suggests Brd4 may regulate microglial activation, proliferation, migration, or viability in tauopathy, in an expression level-dependent manner. Together with mitigation of microgliosis, Tau zebrafish harboring a heterozygous brd4+ / ímutation also showed rescue of synaptic abundance in the optic tectum and dorsal telencephalon. Mutation of the brd4 gene thus phenocopied (+)JQ1-mediated rescue in Tau zebrafish, strongly suggesting that (+)JQ1 exerts its effects in this model by inhibiting Brd4. Studies indicate that the findings in the zebrafish model are applicable to the human CNS. Previous studies showed that BRD4 mRNA is expressed in the human Attorney Docket No.: 49045-0085WO1 / 05762 brain, most prominently the basal ganglia, and enriched in microglia. In a number of studies, human autopsy brain sections from four controls and five PSP cases were labeled with antibodies to Brd4 and Iba1 (a microglial marker). Expression patterns in the substantia nigra and globus pallidus, two areas affected prominently by PSP pathology, were examined. Strong punctate nuclear Brd4 immunoreactivity was found in both quiescent (highly ramified) microglia in control brains, and in activated (retracted processes) microglia in PSP brains. This pattern of nuclear Brd4 expression was found in 1414 / 1432 microglia examined in control brains, and 1648 / 1664 microglia in PSP brains. The data of those studies indicate a phylogenetically conserved role for Brd4 in microglia, supporting the relevance of findings in the zebrafish model to human disease. Figures 2, 3A, 3B, 4A, 4B, 4C, and 4D illustrate results of the screen of 80 kinase inhibitors using methods described herein. Of the 80 compounds studied in the screen of kinase inhibitors at various concentrations, two exceeded the predefined threshold as compounds of interest (Figure 2). In that regard, the compounds staurosporine (Compound #4) and midostaurin (Compound #5) were found to exceed the mean + 3SD threshold. The structures of Compounds #1 through #5 are illustrated in Figure 5. Staurosporine is a natural, alkaloid product that was isolated originally from the bacterium Streptomyces staurosporeus. Midostaurin is a semi-synthetic derivative of staurosporine. Exposure of Tau zebrafish to midostaurin rescued motor function by 73%, while exposure to staurosporine rescued motor function by 60% (Table 2 inset of Figure 2). Figure 4A illustrates representative traces of ocular angle against time for control, Tau zebrafish (Tau), and Tau zebrafish exposed to midostaurin (Tau +). The restorative or rescuing effects of midostaurin are further demonstrated in Figures 4B, 4C, and 4D. Figure 4B illustrates ocular range of movement for 15-20 zebrafish in each group combined from three independent assays. Figure 4C illustrates reflex gain for 15-20 zebrafish in each group combined from three independent assays. Figure 4D illustrates saccade frequency for 15-20 zebrafish in each group combined from three independent assays. Table 3 provides a summary of Compounds #1 - #5, the known function of such compounds, and the target in tauopathy. (+)-JQ1 (Compound #1) is a BET protein inhibitor. Trichostatin A (Compound #2) is a histone deacetylase (HDAC) inhibitor. 2,4- DPD (Compound #3) is a hypoxia-inducible factor prolyl hydroxylase (HIF-PH) Attorney Docket No.: 49045-0085WO1 / 05762 inhibitor. Staurosporine (Compound #4) is an inhibitor of multiple kinases. Midostaurin (Compound #5) is an inhibitor of a more restricted subset of kinases. Only for (+)-JQ1 (Compound #1) has a specific target in tauopathy (that is, Brd4) been determined. That determination is discussed further below. Table 3. Library Chemical Target hy cle in In a number of embodiments of methods of treatment of tauopathy hereof, a pharmaceutical preparation comprising a pharmaceutically effective amount of a compound selected from the group of staurosporine, midostaurin, and biologically active analogs thereof is administered to a patient (e.g., a human patient). The pharmaceutical preparations that contain the staurosporine, midostaurin, and / or biologically active analogs thereof as active ingredients may, for example, be manufactured and administered in a manner similar to pharmaceutical preparations of staurosporine and midostaurin for treatment of other conditions. The pharmaceutical preparations may, for example, be suitable for enteral, such as peroral or rectal, administration, for parenteral administration, and for intrathecal / cerebrospinal fluid (CSF) administration. Corresponding dosage unit forms, especially for peroral administration, for example dragees, tablets or capsules, contain, Attorney Docket No.: 49045-0085WO1 / 05762 for example, from approximately 5 to 500 mg of the active ingredient together with pharmaceutically acceptable carriers or adjuncts. In some embodiments, this document provides methods and materials for treating a tauopathy. Any appropriate mammal having a tauopathy can be treated using the materials and methods described herein. For example, humans or other primates such as monkeys can be identified as having a tauopathy, and a composition (e.g., a pharmaceutical composition) that includes staurosporine, midostaurin, and / or a biologically active analog thereof can be administered to the human or other primate under conditions wherein a neurological function within improved. In some cases, dogs, cats, horses, cows, pigs, sheep, mice, and rats can be identified and treated using the materials and methods described herein. Any appropriate tauopathy can be treated as described herein. For example, progressive supranuclear palsy can be identified in a mammal (e.g., a human) and can be treated by administering a composition (e.g., a pharmaceutical composition) that includes staurosporine, midostaurin, and / or a biologically active analog thereof to that mammal as described herein. Any appropriate composition that includes staurosporine, midostaurin, and / or a biologically active analog thereof can be administered to a mammal (e.g., a human) having a tauopathy to improve a neurological function within the mammal. In some cases, a mammal (e.g., a human) having a tauopathy can be administered or instructed to self-administer a composition that includes staurosporine, midostaurin, and / or a biologically active analog thereof. For example, a mammal (e.g., a human) having a tauopathy can be administered a composition containing midostaurin to improve a neurological function within the mammal. In some cases, when a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof is administered to a mammal (e.g., a human) having a tauopathy, the composition can be designed for oral or parenteral (including, without limitation, subcutaneous, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, intraabdominal, and intraperitoneal injections) administration to the mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the Attorney Docket No.: 49045-0085WO1 / 05762 formulation isotonic with the blood of the intended recipient. In some cases, a pharmaceutically acceptable composition including staurosporine, midostaurin, and / or a biologically active analog thereof can be administered locally or systemically. For example, a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be administered systemically by injection to a mammal (e.g., a human). In some cases, each of staurosporine, midostaurin, and / or a biologically active analog thereof to be administered when two or more agents are to be administered can be administered by the same or different routes. For example, staurosporine can be administered orally, and midostaurin can be administered by injection. In some cases, a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be in the form of a sterile injectable suspension (e.g., a sterile injectable aqueous or oleaginous suspension). This suspension may be formulated using, for example, suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Examples of acceptable vehicles and solvents that can be used include, without limitation, saline, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In some cases, sterile, fixed oils can be used as a solvent or suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides. When staurosporine, midostaurin, and / or a biologically active analog thereof are formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having a tauopathy to improve a neurological function within the mammal as described herein, the composition can deliver the staurosporine, midostaurin, and / or a biologically active analog thereof in any manner. In some cases, a composition including staurosporine, midostaurin, and / or a biologically active analog thereof can be an immediate release composition (e.g., an immediate release oral dosage form). In some cases, a composition including staurosporine, midostaurin, and / or a biologically active analog thereof can be a controlled (e.g., delayed and / or sustained) release compositions (e.g., a controlled release oral dosage form). For example, a controlled release composition including staurosporine, midostaurin, and / or a biologically active analog thereof can be designed to release the staurosporine, midostaurin, and / or a biologically active analog thereof over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Attorney Docket No.: 49045-0085WO1 / 05762 days. When designing a composition (e.g., a pharmaceutically acceptable composition) containing staurosporine, midostaurin, and / or a biologically active analog thereof for administration to a mammal (e.g., a human) having a tauopathy to improve a neurological function within the mammal as described herein, the composition (e.g., pharmaceutically acceptable composition) can include any appropriate effective amount of staurosporine, midostaurin, and / or a biologically active analog thereof. For example, a therapeutically effective amount of staurosporine, midostaurin, and / or a biologically active analog thereof can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, cyclodextrins (e.g., beta-cyclodextrins such as KLEPTOSE®), dimethylsulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene- polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline such as phosphate buffered saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil. If required, the solubility and bioavailability of staurosporine, midostaurin, and / or a biologically active analog thereof in a pharmaceutically acceptable composition can be enhanced using lipid excipients and / or block copolymers of ethylene oxide and propylene oxide. See, e.g., U.S. Patent No. 7,014,866 and U.S. Patent Application Publication Nos. 2006 / 0094744 and 2006 / 0079502. Attorney Docket No.: 49045-0085WO1 / 05762 In some cases, a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be designed to be in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Staurosporine, midostaurin, and / or a biologically active analog thereof (e.g., a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof) can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to improve a neurological function within a mammal having a tauopathy). An effective amount of staurosporine, midostaurin, and / or a biologically active analog thereof that can be administered to a mammal as described herein can be any amount that improves a neurological function within a mammal having a tauopathy without producing significant toxicity to the mammal. For example, an effective amount of staurosporine, midostaurin, and / or a biologically active analog thereof can be from about 0.01 mg of staurosporine, midostaurin, or a biologically active analog thereof / kg of body weight to about 80 mg of staurosporine, midostaurin, or a biologically active analog thereof / kg of body weight (e.g., from about 0.1 mg / kg to about 70 mg / kg, from about 0.1 mg / kg to about 60 mg / kg, from about 0.1 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.1 mg / kg to about 30 mg / kg, from about 0.1 mg / kg to about 20 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.075 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, or from about 2 mg / kg to about 10 mg / kg). In some cases, between about 1 mg and 200 mg (e.g., between about 50 mg and 180 mg, between about 50 mg and 150 mg, between about 50 mg and 125 mg, between about 50 mg and 100 mg, between about 60 mg and 200 mg, between about 75 mg and 200 mg, between about 100 mg and 200 mg, between about 75 mg and 150 mg, or between about 100 mg and 150 mg) of staurosporine, midostaurin, or a biologically active analog thereof can be administered to an average sized human (e.g., about 70 kg human) daily for about 20 weeks. If a particular mammal fails to respond to a particular amount, then the amount of staurosporine, midostaurin, and / or a biologically active analog thereof can be increased by, for example, two fold. After receiving this higher amount, the mammal can be monitored for both responsiveness to the treatment Attorney Docket No.: 49045-0085WO1 / 05762 and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition can require an increase or decrease in the actual effective amount administered. A composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be administered to a mammal (e.g., a human) having a tauopathy in any appropriate frequency. The frequency of administration can be any frequency that improves a neurological function within a mammal having a tauopathy without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about three times a day, or from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can include rest periods. For example, a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be administered daily over a two week period followed by a two week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition can require an increase or decrease in administration frequency. A composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be administered to a mammal (e.g., a human) having a tauopathy for any appropriate duration. An effective duration for administering a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can be any duration that improves a neurological function within a mammal having a tauopathy without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several weeks, months, or years. In general, the effective duration for the treatment of a tauopathy as described herein can range in duration from several months to several years. In some cases, an effective duration can Attorney Docket No.: 49045-0085WO1 / 05762 be for as long as an individual mammal is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated. In some cases, methods for treating a mammal (e.g., a human) having a tauopathy as described herein (e.g., by administering staurosporine, midostaurin, and / or a biologically active analog thereof) can include administering to the mammal the staurosporine, midostaurin, and / or a biologically active analog thereof as the sole active ingredient(s) to treat a tauopathy within the mammal. For example, a composition containing staurosporine, midostaurin, and / or a biologically active analog thereof can include the staurosporine, midostaurin, and / or a biologically active analog thereof as the sole active ingredient in the composition that is effective to improve a neurological function within a mammal having a tauopathy. In some cases, a course of treatment and the level of neurological functions within a mammal can be monitored. Any appropriate method can be used to determine whether or not treatment with staurosporine, midostaurin, and / or a biologically active analog thereof as described herein is effective to improve a neurological function within a mammal having a tauopathy. For example, neurological functions during a course of treatment described herein can be assessed using imaging techniques at different time points. The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES Example 1 – Identifying agents for treating a tauopathy Abstract Attorney Docket No.: 49045-0085WO1 / 05762 Progressive supranuclear palsy (PSP) is an incurable neurodegenerative disease characterized by accumulation of 4-repeat Tau protein (0N / 4R-Tau) in CNS neurons. We generated transgenic zebrafish expressing human 0N / 4R-Tau to investigate PSP pathophysiology. Tau zebrafish replicated multiple features of PSP, including decreased survival, hypokinesia, impaired optokinetic responses, neurodegeneration, neuroinflammation, synapse loss, and Tau hyperphosphorylation, misfolding, mislocalization, insolubility, and oligomerization. Using automated motor function assays, we screened a chemical library for compounds that rescued neurological deficits in Tau zebrafish. (+)JQ1, a bromodomain inhibitor, mitigated motor function, survival, neuroinflammation, and brain synapse elimination. A brd4 mutant similarly rescued these phenotypes, confirming that (+)JQ1 acts by targeting Brd4 in this model. Microglia in human PSP brains also expressed Brd4, suggesting a phylogenetically-conserved role in CNS innate immunity. Our work implicates Brd4 as a regulator of microglial synaptic elimination in neurodegeneration and provides a new approach for identifying molecular mechanisms and therapeutic targets in PSP. Introduction Progressive supranuclear palsy (PSP) is a sporadic neurodegenerative disease characterized by falls, hypokinesia, dysphagia, oculomotor deficits and dementia1. Median onset is age 672, median survival is 8 years3, and there are no effective treatments. Pathologically, neuronal loss in brainstem and basal ganglia nuclei is accompanied by microglial activation and formation of neuronal intracellular inclusions composed of insoluble, hyperphosphorylated aggregates of the microtubule-associated protein Tau4. Accumulation of Tau protein in PSP predominantly involves isoforms containing 4 microtubule-binding domain repeats and lacking N-terminal insertions (0N / 4R-Tau)5,6(this contrasts with Alzheimer’s disease and chronic traumatic encephalopathy, in which multiple 3R- and 4R-Tau isoforms are deposited). Genetic evidence supporting a causative role for 4R-Tau in PSP includes: clinical-pathologic PSP phenocopies caused by mutations in the MAPT gene encoding Tau7; a locus inversion allele encompassing the MAPT gene that expresses less 4R-Tau8and is protective against PSP9; and a strong association between non-coding SNPs near to MAPT and PSP risk10,11. However, interventions targeting Tau directly did not prevent progression of PSP in clinical trials12-14. This suggests mechanisms downstream of 4R-Tau accumulation may Attorney Docket No.: 49045-0085WO1 / 05762 provide more appropriate therapeutic targets once the disease is sufficiently advanced to cause diagnostic clinical deficits. Unbiased discovery-driven approaches such as chemical modifier screens may help elucidate the molecular pathophysiology of PSP, particularly as the downstream consequences of 4R-Tau accumulation in the nervous system are incompletely understood. In addition, many first-in-class drugs were discovered by phenotypic screening15, an important consideration given the lack of therapeutic options for PSP patients. An unbiased chemical screen would require a high-throughput model that replicates the complexities of PSP pathophysiology – which occurs in end-differentiated neurons, involves cell-autonomous and non-autonomous events, and expresses clinical endpoints by disrupting the functions of neural circuits. These considerations suggest that a screen should be completed in vivo to capture the relevant pathogenic events. Zebrafish show substantial genetic and neurological homology to humans and, uniquely amongst vertebrates, offer opportunities for chemical modifier screening in vivo16,17. Notably, screening in a zebrafish PSP model might employ automated assays of disease-relevant neurological phenotypes as readouts, in order to identify chemical modifiers without bias concerning mechanism of action. However, although there has been significant interest in the development of transgenic zebrafish expressing human Tau for this purpose18, most of the reported lines express mutated forms of human Tau that are not present in PSP19,20, and none of the current models has a PSP-like phenotype that is sufficiently robust for chemical screening19-23. Results Zebrafish model of progressive supranuclear palsy To develop a zebrafish PSP model optimized for chemical screening applications, we employed Gal4-UAS genetics (Figure 6A, Figure 12). The UAS responder cassette encodes WT human 0N / 4RTau, together with nls-mCherry to allow rapid identification of transgenic zebrafish by fluorescence microscopy in downstream applications. The 2A peptide24ensures that human 0N / 4R-Tau and nls-mCherry are expressed as separate proteins (directed to different subcellular compartments; Figure 6B) from the same mRNA transcript, thereby avoiding the potential for a fluorescent fusion protein to alter the pathophysiological properties of Tau. By generating Tg(UAS:hsa.MAPT-p2A-nls- mCherry) lines in the absence of Gal4-induced expression, we isolated stable heritable Attorney Docket No.: 49045-0085WO1 / 05762 transgene alleles that can be propagated readily. We then selected lines that showed robust transactivation when crossed with a pan-neuronal Gal4 driver25to yield Tg(elavl3:Gal4-VP16); Tg(UAS:hsa.MAPT-p2A-nls-mCherry) ‘Tau’ zebrafish. Tg(elavl3:Gal4-VP16); Tg(UAS:p2A-nls-mCherry) ‘Ctrl’ zebrafish were generated to provide controls with the same complement of transgenes as Tau zebrafish but lacking human 0N / 4R-Tau (Table 4). Table 4 – Abbreviations and genotypes of zebrafish lines used in this study The lifespan of Tau zebrafish (median survival 8 – 9 days in replicate cohorts) was severely attenuated in comparison with Ctrl zebrafish or non-expressing siblings (both control lines survive to aged adulthood, >2 years; Figure 6C, D; Figure 13; Table 5). Three complementary assays – failure of acridine orange exclusion in vivo (Figure 6E), TUNEL labeling of apoptotic cells in histological sections (Figure 6F), and detection of Capsase-3 cleavage (both in sections and by western blot; Figure 6G) – demonstrated a robust increase in neuronal death in Tau compared with Ctrl zebrafish between 2 – 7 days post-fertilization (dpf; Figures 14 and 15). Peak cell death was detected in Tau zebrafish in all three assays at 3 – 4 dpf, with up to 10-fold excess apoptotic cells compared with Ctrl. By 5dpf, decreases in the expression of dopaminergic (Tyrosine Hydroxylase; TH) and GABAergic (Glutamic Acid Decarboxylase; GAD) neuronal markers, and in both pre- (Synaptophysin; SYP) and post- (Post-Synaptic Density 95 kDa; PSD95) synaptic markers, were apparent in Tau zebrafish compared with Ctrl by western blot (Figure 6H). Immunohistochemistry also showed a large (> 2-fold) increase in brain microglia in Tau zebrafish compared with Ctrl between 3 – 6 dpf (Figure 6J). Together, these data show that conditional expression of human 0N / 4R-Tau in transgenic zebrafish causes impaired survival, neurodegeneration, loss of neurochemical and synaptic markers, and neuroinflammation, which are key features of PSP. Attorney Docket No.: 49045-0085WO1 / 05762 Table 5 – Statistical analysis of survival in Tau zebrafish Human 0N / 4R-Tau from Tau zebrafish at 5dpf migrated on western blot at 64kDa (Figure 7A), near the observed electrophoretic mobility of hyperphosphorylated 0N / 4R- Tau in human PSP brain5,6. Dephosphorylation of Tau zebrafish lysate decreased the apparent molecular mass of 0N / 4R-Tau to 52kDa and eliminated immunoreactivity to a human phospho-Tau-specific antibody, suggesting that human Tau becomes hyperphosphorylated in the zebrafish CNS (Figure 7B). Consistent with this interpretation, a 64kDa band in zebrafish lysate was immunoreactive to six different antibodies that detect phospho-Tau epitopes distributed across the proline-rich and C- terminal domains of the human protein (Figure 7C and 7D)26. Hyperphosphorylated human 4R / 0N-Tau was detected in the cell bodies, proximal processes, and axons of neurons throughout the CNS of Tau zebrafish (Figure 7E). Some cells were also labeled by antibodies detecting epitopes specific to misfolded Tau (Alz50, MC1; Figure 7E and 7F)27, or caspase-cleaved Tau (TauC3; Figure 7G; truncated Tau fragments were also apparent by western blot, Figure 16)28found in human tauopathies. Between 3 – 6 dpf, total and phosphorylated human Tau in RIPA (1% Triton, 0.1% SDS)-extracted lysates declined steadily; however, no such decrease was noted in DIGE (7M urea)-extracted lysates, suggesting progressive loss of 0N / 4R-Tau solubility in the zebrafish brain (Figure 7H). Non-denaturing gel electrophoresis demonstrated the presence of higher molecular weight forms, corresponding to oligomers of truncated Tau (Figure 7J). Together these data show that human 0N / 4R-Tau in the zebrafish brain becomes hyperphosphorylated, mislocalized, misfolded, truncated, loses solubility, and becomes oligomerized, replicating many features of human tauopathies including PSP. We did not find evidence of large argyrophilic amyloid aggregates resembling neurofibrillary tangles over the short Attorney Docket No.: 49045-0085WO1 / 05762 time course of these experiments. This is similar to other rapidly progressive tauopathy models29, consistent with prominent pre-tangle pathology found in PSP4, and possibly related to the relative infrequency of cells immunoreactive for caspase-cleaved Tau, which may be a precursor of larger aggregates30. Automated machine vision measurement of zebrafish motor activity in 96-well plates at 5dpf (Figure 8A)31-33showed that Tau zebrafish were profoundly hypokinetic in comparison with Ctrl zebrafish during spontaneous movement in bright ambient illumination over prolonged recordings (Figure 8B; Table 6). Mean scalar speed (centroid displacement / time) is an integrative measurement of zebrafish motility, reflecting both the speed and size of individual movements and the frequency with which they are executed. A robust decrease in mean speed in Tau zebrafish compared with Ctrl zebrafish was observed in multiple replicate assays (Figure 8C; Figure 17; Tables 7 and 8) and was attributable to: (i) a large increase in the time interval between movements, combined with a small decrease in the duration of movement events, resulting in a decreased proportion of the assay during which Tau zebrafish were motile; and (ii) a modest decrease in the speed of individual movements by Tau zebrafish (Figures 8D, 8E, and 8F; Figures 18 and 19). High temporal and spatial resolution kinematic analysis of motor responses evoked by abrupt ambient light-dark transition (Figure 8G)34showed that peak truncal curvature and peak truncal angular velocity were unchanged in Tau zebrafish compared with Ctrl during high-angle ‘O’-bend turning movements, even though the response rate to illumination transitions was lower and the latency to response prolonged (Figure 8H; Figure 20). These data suggest that hypokinesia in Tau zebrafish is not attributable to neuromuscular paralysis, but is caused by reduced movement initiations, with possible parallels to the akinetic motor disorder seen in PSP patients.

[0002] Attorney Docket No.: 49045-0085WO1 / 05762 Table 6 – Šidák multiple comparisons test for Figure 8B

[0003] Attorney Docket No.: 49045-0085WO1 / 05762 Table 7 – ANOVA summary for Figure 17 Table 8 - Šidák multiple comparisons test for Figure 17 In view of the prominent oculomotor disorder for which PSP is named, we next evaluated the eye movements of Tau zebrafish by analyzing optokinetic reflexes35,36. Projection of an animated grating pattern on a screen filling the zebrafish visual field provokes characteristic cycles of slow ocular movements in the stimulus direction (that serve to stabilize the moving image in the retina) and rapid positional resetting movements (saccades) in the opposite direction (Figure 8Jand 8K; and Figure 21). Compared with Ctrl zebrafish, Tau zebrafish showed a decreased range of ocular movement, reflex tracking gain, and saccade frequency, in addition to disrupted coordination between movements of the two eyes (Figure 8K – 8N, Figure 21D). Complete loss of saccades during OKR was observed in 60% Tau zebrafish, resembling a characteristic clinical abnormality found in PSP patients37. Overall, these data show that Tau zebrafish develop robust OKR deficits reminiscent of those found in PSP. Phenotype-based chemical modifier screen in vivo Attorney Docket No.: 49045-0085WO1 / 05762 We next investigated whether automated measurement of hypokinesia could be used as an unbiased and quantitative screening endpoint to detect interventions that rescue the neurological phenotype of Tau zebrafish. For these analyses, we compared Tau zebrafish with non-expressing siblings, to simplify breeding for subsequent screening. This approach is valid, as Ctrl zebrafish and non-expressing siblings of Tau zebrafish showed identical responses in 96-well plate motor assays (Figure 19). Variability in measured motor activity was minimized by averaging the responses to multiple cycles of alternating ambient illumination and darkness (Figure 22; Table 9). Mean light phase swimming speed showed the largest and most replicable difference between Tau zebrafish and their siblings in these assays, but analysis of single zebrafish using this metric as an endpoint did not provide adequate performance as a screening assay (Figure 9A). However, similar to our previous findings in chemically-induced parkinsonism models32, averaging the responses of multiple zebrafish in each treatment group substantially improved assay performance at the expense of throughput (Figure 9B). The optimal group size of 12 Tau zebrafish allowed 6 compounds to be tested in parallel with controls in each 96-well plate, with a consistently positive Z’ indicating a remarkable level of performance for a neurobehavioral assay (Figure 9A and 9B).

[0004] Attorney Docket No.: 49045-0085WO1 / 05762 Table 9 – Šidák multiple comparisons table for Figure 22 We selected a library of 140 inhibitors of epigenetic readers, writers, and erasers, because its compounds target interesting cellular physiology that is relatively unexplored in tauopathy, and the modest library size is manageable for an initial screen against the Tau model. Screening was completed in two phases: (i) the maximum tolerated concentration (MTC) of each chemical was defined experimentally in WT zebrafish using survival and morphological endpoints (Figure 23); (ii) each chemical was then tested at MTC in 12 Tau zebrafish, and their mean light phase swimming speed normalized to the Attorney Docket No.: 49045-0085WO1 / 05762 phenotypic window defined by 12 untreated Tau zebrafish and 12 non-expressing siblings in each assay (Figure 9C; Figure 24). These controls provided stringent inbuilt assay QC, and normalization of chemical responses to within-assay controls allowed comparison between assays. Both QC benchmarks and data analysis were automated to eliminate bias and accelerate workflow (Figures 25 and 26), and chemicals were screened with their identities masked until the entire library had been analyzed. Mean ± SE % phenotypic rescue for each compound is shown in Figure 9D in comparison with untreated Tau zebrafish (0% rescue) and non-expressing siblings (100% rescue; numerical data, chemical identities, and targets are shown in Table 10). Three of the compounds satisfied our a priori definition of a ‘hit’ (mean library rescue + 3 SD; Figure 9D, yellow data points, and inset table 4D’; Figure 27), whereas the remainder were distributed around the untreated Tau group.

[0005] Attorney Docket No.: 49045-0085WO1 / 05762 Table 10 – Summary of data from the chemical screen. Summary table of data from chemical screen. Rows show each library chemical, ranked by % rescue of the light phase of the VMR; hits shown in red, other compounds of potential interest shown in green. Columns (from left to right): rank; maximum tolerated concentration (i.e. concentration tested in this assay; ^M); mean % rescue of the light phase of the VMR (negative values indicate chemical worsened the motor phenotype of Tau zebrafish); SE of rescue; chemical identity; chemical target.

[0006] Attorney Docket No.: 49045-0085WO1 / 05762

[0007] Attorney Docket No.: 49045-0085WO1 / 05762

[0008] Attorney Docket No.: 49045-0085WO1 / 05762 The top hit, (+)JQ1, disrupts binding of acetylated lysine residues to the bromodomains of bromo- and extraterminal-domain containing (BET) proteins, which regulate transcriptional responses to histone acetylation. The other two hits were Trichostatin-A (inhibits multiple histone deacetylases, HDACs) and 2,4-DPD (inhibits hypoxia-inducible factor-1Į prolyl hydroxylase). Clustering the compounds by pharmacological target showed that BET, multi-HDAC, or DNA methyltransferase inhibitors overall improved motor function significantly (Figure 9E; Figure 28, Tables 10, 11, and 12). There was substantial variability between compounds within each target class that likely reflects variation in their pharmacokinetics or differential binding affinity for zebrafish proteins38. Repurchased (+)JQ1 rescued Tau motor function in a replicable, concentration-dependent manner, formally confirming its identity (Figures 9F, 10A; Figure 29). The inactive stereoisomer (í)JQ1 did not rescue motor function in Tau zebrafish (Figure 9D green data point, and Figure 10B) demonstrating the requirement of a specific ligand-bromodomain interaction for activity. (+)JQ1 did not increase the swimming speed of Ctrl zebrafish (Figure 10C), excluding non-specific stimulation of zebrafish motility as the explanation for our observations. Exposure to (+)JQ1 did not Attorney Docket No.: 49045-0085WO1 / 05762 reduce 0N / 4R-Tau levels (Figure 10D), alter Tau phosphorylation (determined by electrophoretic mobility or immunoreactivity to phosphoepitope-specific antibodies), or silence transgene expression (Figure 10E and 10F). Together these data suggest that (+)JQ1 acts to inhibit pathophysiology in this model conceptually downstream of Tau. Table 11 – Data summary by target Attorney Docket No.: 49045-0085WO1 / 05762 Table 12 – Properties of chemicals categorized as ‘other’ in Figure 9E (+)JQ1 targets Brd4 to inhibit microgliosis and synapse elimination in Tau zebrafish Median survival of Tau zebrafish was increased 15 – 20% by exposure to (+)JQ1, which did not alter survival of Ctrl zebrafish (Figure 10G – 10J; Figures 30, 31; Tables 13 and 14). Interestingly, (+)JQ1 did not decrease acridine orange labeled cells (Figure 10K), suggesting that hypokinesia is not caused by spinal cord cell death in this model. Importantly, however, (+)JQ1, but not (í)JQ1, decreased CNS microglial abundance substantially in Tau zebrafish at 5dpf (Figure 10L, and 10M; decreased removal of dead cells by phagocytosis may account for their modest increase in Tau zebrafish exposed to (+)JQ1 in Figure 10K). Microglial synaptic pruning was identified recently as an important mechanism in neurodegenerative disease models39and synaptic loss was previously demonstrated pathologically in PSP40,41. Since (+)JQ1 prevents microgliosis in Tau zebrafish, we next asked whether improvement of motor function might be explained by (+)JQ1 rescuing synapse elimination. Western blot analysis showed that exposure to (+)JQ1 restored expression of the post-synaptic marker PSD95 in Tau zebrafish (Figure 10N). Volumetric imaging in whole mount brains, coupled with unbiased automated image analysis (Figure 10P; Figure 32), showed that the density of PSD-95 immunoreactive synaptic puncta was decreased in both the optic tectum (Figure 10Q) and Attorney Docket No.: 49045-0085WO1 / 05762 dorsal telencephalon (Figure 10R) of Tau zebrafish compared with Ctrl, but this was rescued by exposure to (+)JQ1. These data together suggest that (+)JQ1 improves motor function and survival in Tau zebrafish by preventing microglial elimination of synapses. Intriguingly, several phenotypic components of Tau zebrafish, including decreased motor activity in the dark phase of the VMR and disrupted OKRs in Tau zebrafish, were not rescued by (+)JQ1 (Figure 33); these may be mediated by a different mechanism. Table 13 – Statistical analysis of Tau zebrafish survival in presence of absence of (+)JQ1 Table 14 – Statistical analysis of Ctrl zebrafish survival in presence of absence of (+)JQ1 Loss of Brd4 phenocopies the effects of (+)JQ1 in Tau zebrafish We verified the molecular target of (+)JQ1 in the zebrafish Tau model using reverse genetics. Previous work showed that (+)JQ1 binds with high affinity to the BET protein Brd442, which has been implicated in inflammatory signaling in other contexts43. Zebrafish have a single highly-conserved orthologue of human Brd444. Antisense morpholino oligonucleotides (MO) that target splicing or translation of the zebrafish brd4 mRNA decreased Brd4 expression and mitigated microgliosis in Tau zebrafish at 3 – 4dpf (Figure 11A; Figure 34). However, progressive loss of MO during cell division following microinjection means that gene knockdown only lasts until 2 – 3 dpf16. In order to test the role of Brd4 in Tau zebrafish at later timepoints when phenotypic abnormalities are most prominent, we engineered a stable 11-bp deletion in exon 4 of brd4 that abrogated Brd4 Attorney Docket No.: 49045-0085WO1 / 05762 expression (allele designation Pt435), using Cas9 / CRISPR (Figure 11A, and 11B; Figure 35). Homozygous brd4í / í mutants lacking Brd4 showed morphological and motor deficits, and were not viable (Figures 36, 37). However, heterozygous brd4+ / í zebrafishshowed no overt abnormalities, and [Ctrl; brd4+ / í] zebrafishshowed similar motorfunction to their [Ctrl; brd4+ / +] siblings (Figure 11D), allowing us to test the effect of the heterozygous brd4 mutation on the neurological phenotypes of Tau zebrafish. The VMR light phase mean speed of [Tau; brd4+ / í] zebrafish was significantly higher than [Tau; brd4+ / +] siblings, demonstrating that decreased Brd4 expression partially rescued hypokinesia in Tau zebrafish (Figure 11C, 11D; Figure 38). Similar to (+)JQ1 exposure, reduced Brd4 expression did not rescue acridine orange labeled spinal cord cells or OKR deficits in Tau zebrafish (Figure11; Figure 39). However, there was a robust decrease in CNS microglial abundance in zebrafish harboring brd4 mutations (Figure 11F). The density of brain microglia in Tau zebrafish was highest in brd4+ / + zebrafish, decreased in brd4+ / í heterozygotes, and lowest in homozygous brd4í / í mutants. These data suggest Brd4 may regulate microglial activation, proliferation, migration, or viability in tauopathy, in an expression level-dependent manner. Together with mitigation of microgliosis, Tau zebrafish harboring a heterozygous brd4+ / í mutation also showed rescue of synaptic abundance in the optic tectum and dorsal telencephalon (Figure 11G, 11H; Figure 40). Mutation of the brd4 gene thus phenocopied (+)JQ1-mediated rescue in Tau zebrafish, strongly suggesting that (+)JQ1 exerts its effects in this model by inhibiting Brd4. Brd4 is expressed in human microglia Finally, we asked whether our findings in the zebrafish model might be applicable to the human CNS. Prior work showed that BRD4 mRNA is expressed in the human brain, most prominently the basal ganglia, and enriched in microglia45. We labeled human autopsy brain sections from 4 controls and 5 PSP cases with antibodies to Brd4 and Iba1 (a microglial marker). We examined expression patterns in the substantia nigra and globus pallidus, two areas affected prominently by PSP pathology1,4. Strong punctate nuclear Brd4 immunoreactivity was found in both quiescent (highly ramified) microglia in control brains, and in activated (retracted processes) microglia in PSP brains (Figure 11J, Figure 41). This pattern of nuclear Brd4 expression was found in 1414 / 1432 microglia examined in control brains, and 1648 / 1664 microglia in PSP brains (Figure Attorney Docket No.: 49045-0085WO1 / 05762 11K; Figure 42). These data suggest a phylogenetically conserved role for Brd4 in microglia, supporting the relevance of our findings in the zebrafish model to human disease. Discussion Development of a zebrafish PSP model suitable for unbiased phenotype-driven chemical screening is a significant advance. By generating UAS responder zebrafish in the absence of Gal4 (a laborious approach owing to the lack of a fluorescent marker), we avoided negative selection against larvae with impaired viability attributable to 4R-Tau expression. Selection for responder lines with strong expression after crossing with a pan- neuronal Gal4 driver yielded Tau zebrafish with robust PSP-relevant phenotypes at larval timepoints when screening is practicable. Importantly, the model has construct validity, as its phenotypes are caused by the same WT human 0N / 4R-Tau isoform that accumulates in PSP5,6. The model also has strong face validity since it replicates many of the core clinical (hypokinesia, oculomotor deficits, impaired survival), pathological (neurodegeneration, neuroinflammation) and biochemical (Tau hyperphosphorylation, mislocalization, misfolding, truncation, insolubility, and oligomerization) features of PSP. We showed proof of concept that phenotypic screening in a zebrafish tauopathy model can identify chemical modifiers that yield insights into pathophysiology. Our work represents the first time this idea has been realized successfully in practice. The use of PSP-relevant neurological deficits as endpoints – made possible by the robust phenotype of the model, coupled with optimized assay workflows32– allowed analysis of molecules with a broad range of activities that were not limited by preconceptions about mechanism of action. This resulted in unbiased identification of several pathways potentially involved in 4R-tauopathy pathogenesis. Histone acetylation46and HIF-1Į signaling47were previously implicated in other neurodegenerative diseases, but have not been investigated in PSP. Our data showing that DNA methyltransferase inhibitors improved phenotypes in Tau zebrafish are compatible with previous work showing DNA hypermethylation at multiple gene loci in PSP brains48. Together, these observations provide preliminary evidence that Tau zebrafish may show predictive validity as a PSP model and suggest additional directions for future investigation. However, although unbiased, our screen was limited in scope by the modest size and focused nature of the chemical library. Given the rich yield from this initial screen, we are currently scaling the Attorney Docket No.: 49045-0085WO1 / 05762 approach to larger libraries encompassing more diverse molecular targets. We anticipate that the model, together with the open-source applications we developed for its automated quantitative analysis31,33,36, will be useful for additional chemical biology applications, including optimizing experimental PSP therapeutics rapidly in vivo, and genetic approaches (for which zebrafish are especially suited), such as analysis of pathophysiological mechanisms and PSP risk genes, and verification of chemical modifier targets, as demonstrated here for (+)JQ1 and Brd4. Identification of Brd4 as a contributor to synapse elimination in tauopathy highlights the value of screening in vivo, as the cell non-autonomous mechanism requires both microglia and neural circuits with functional synaptic connections, which would be difficult to replicate in an in vitro screen. Microglial synaptic elimination strongly influenced motor function and survival in Tau zebrafish, underlining its significance in driving phenotypic outcomes in this model. Recent work showed that synaptic loss in mouse Alzheimer disease models involves deposition of complement C1q and C3 at synapses, and complement-dependent microglial engulfment activated via C3R receptors39,49,50. Complement also plays a key role in primary tauopathy models, in which synaptic loss was rescued by C1q antibodies51or loss of C3aR1 receptors52, and negatively regulated by Nptx2, a secreted neuronal pentraxin that binds C1q53. It is currently unclear how Brd4 regulates this process. A BET inhibitor was recently reported to downregulate basal and cytokine-simulated expression of complement components in cultured hepatocytes and humanized mice, and to reduce activated complement C3 and C5 levels in the circulation of cardiovascular disease patients54, but the role of Brd4 in CNS complement functions is currently unknown. Alternatively, our observations might be attributable to changes in microglial abundance or other non-complement functions that have also been implicated in tauopathy progression55,56. Tau zebrafish provide a tractable experimental system to test these possibilities systematically, facilitated by the extensive phylogenetic conservation of complement57and microglial transcriptional programs58between zebrafish and mammals, and by the ability to monitor microglial behavior directly in transgenic zebrafish by intravital microscopy59. The improvement of survival and neurological function we observed in Tau zebrafish exposed to (+)JQ1 suggests that the translational potential of bromodomain inhibitors in PSP should be explored further, especially as compounds targeting BET bromodomains have already been tested in clinical studies for other indications (although Attorney Docket No.: 49045-0085WO1 / 05762 they caused some toxicities)60,61. The expression of Brd4 in human microglia is encouraging that our findings may be relevant to PSP, and prior work supports the premise that bromodomain inhibitors mitigate neuroinflammation62-64. (+)JQ1 was previously shown to reduce inflammatory marker expression in the CNS of a mouse Alzheimer’s disease model, but did not rescue neurological phenotypes63, possibly owing to its short half-life (0.9 hours)62coupled with intermittent administration (this is not a concern in zebrafish, which are bathed continuously in a large volumetric excess of (+)JQ1 solution). However, the phenotypes of Tau zebrafish were not rescued completely by either (+)JQ1 or decreased Brd4 expression; while this may reflect the timing or potency of the experimental manipulations relative to pathogenesis in the model, these data also raise the intriguing possibility that specific components of the full phenotypic profile are driven by distinct molecular mechanisms. Functional imaging studies in patients confirm that microglial activation65and synaptic loss66occur during PSP pathogenesis. It will be important to understand how these, and other mechanisms, contribute to the progression of neurological deficits in PSP, as this will strongly influence the translational utility of any interventions targeting microglial synaptic elimination. Methods DNA constructs A 2A-nls DNA fragment was amplified from plasmid T2M-mCherry-2A-nls- eGFP (obtained from Dr. Steven Leach, Dartmouth College)24using primers 5ƍ- CGGGATCCGGAGCCACGAAC-3ƍ (SEQ ID NO:13) and 5’- CCATGTTATCCTCCTCGCCCTTGCTCAC-3ƍ (SEQ ID NO:14), digested with BamHI, and inserted into the BamHI / MscI sites of pmCherry (Clontech, Palo Alto, CA) to make p2A-nls-mCherry. A 2A-nls-mCherry fragment was then amplified from p2A-nls- mCherry using primers 5ƍ GTGATATCCGGAGCCACGAACTTC-3ƍ (SEQ ID NO:15) and 5ƍ- CCGCTCGAGCCGCTACTTGTACAGC-3ƍ (SEQ ID NO:16), digested with EcoRV and XhoI and inserted into pT2MUASMCS (obtained from Dr. Koichi Kawakami, National Institute of Genetics, Japan)67to make pTol2-5UAS:2A-nlsmCherry. The open reading frame of human 4R / 0N-Tau was amplified from plasmid t2 (obtained from Dr. Matthew Farrer, University of Florida)21using primers 5ƍ- GAAGATCTGTCGACGAATTCCC-3ƍ (SEQ ID NO:17) and 5ƍ- Attorney Docket No.: 49045-0085WO1 / 05762 CCCAAACCCTGCTTGGCCAG-3ƍ (SEQ ID NO:18), digested with BglII and inserted into the BglII / EcoRV sites of pTol2- 5UAS:2A-nls-mCherry to make pTol2- 5UAS:hsa.MAPT-2A-nls-mCherry. All DNA constructs were sequenced fully, and expression of mCherry was verified in transient co-transfection experiments prior to generating transgenic lines. Transgenic zebrafish Stable transgenic zebrafish lines were generated by modification of procedures described in our previous work, by microinjecting pTol2-5UAS:2A-nls-mCherry or pTol2-5UAS:hsa.MAPT-2A-nls-mCherry, together with mRNA encoding Tol2 transposase, into single-cell embryos from WT strain AB zebrafish68,69. For pTol2- 5UAS:hsa.MAPT-2A-nls-mCherry, we identified 10 different F0 chimeras by PCR of pooled F1 embryo DNA resulting from a WT outcross, using primers 5ƍ- AGATCTGTCGACGAATTCCC-3ƍ (SEQ ID NO:19) and 5ƍ- AATCCTGGTGGCGTTGGCCT-3ƍ (SEQ ID NO:20). mCherry expression was then evaluated in the F1 progeny of each F0 chimera, after crossing with a Tg(eno2:Gal4FF) neuronal driver line70. Four of the crosses generated progeny with bright fluorescence; the F0 parent in each cross was then crossed again with WT zebrafish, and the progeny raised to adulthood. F1 founders for each Tg(UAS:hsa.MAPT-2A-nls-mCherry) line were identified by PCR of fin clip DNA from adults. mCherry expression was verified in F2 progeny of F1 x Tg(eno2:Gal4FF) crosses. Lines were then derived from single F1 founders. Derivation, expansion, and maintenance of lines was carried out by WT outcross, in the absence of a Gal4 driver, using fin clip PCR genotyping. Of several Gal4 pan-neuronal driver lines tested, Tg(elavl3:gal4-vp16)25yielded the strongest transactivation of the UAS alleles and was used for subsequent experiments. Identical phenotypes were observed in multiple different 4R-Tau responder alleles when crossed to the pan-neuronal driver. Line Tg(UAS:hsa.MAPT-2A-nls-mCherry)Pt433 showed stable expression and minimal variegation over multiple generations (currently F9) and was used for the studies shown here. Tg(UAS:2A-nls-mCherry)Pt434 controls were generated similarly but can be maintained on a Gal4 driver background as they have no phenotype other than neuronal mCherry fluorescence. Brd4 knockdown with morpholino oligonucleotides Attorney Docket No.: 49045-0085WO1 / 05762 Custom morpholino oligonucleotides (MO; Gene Tools, Philomath, OR) were designed to target the translational start site of brd4 mRNA 5ƍ- CGTCCAGGCCGTCCCCCATACTAG-3ƍ (SEQ ID NO:21) or the slice acceptor at the intron 5 / exon 6 boundary in the primary brd4 transcript 5ƍ- TCATGTCTAATGACACAGAAAGAGA-3ƍ (SEQ ID NO:22). MO stocks were diluted to a working concentration of 5 ng / nL in Danieau microinjection buffer (8 mM NaCl, 0.7mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6) with 0.5% phenol red.1 to 2nL of MO solution was injected into the yoke sac of each embryo at 1 – 4 cell stage. A standard nontargeting MO 5ƍ-CCTCTTACCTCAGTTACAATTTATA-3ƍ (SEQ ID NO:23; cat # PCO-StandardControl-100; Gene Tools) was used as a negative control at the same concentration and volume. Microinjected embryos were allowed to develop to 2 – 4 dpf then lysed in RIPA for Western blot (see below) or fixed for immunohistochemistry to quantify microglia. Brd4 knockout zebrafish A custom gRNA 5ƍ-GCCGGGGCAGGAGGGAUCC-3ƍ (SEQ ID NO:24) was designed to target a BamHI restriction site at the 3’ end of zebrafish brd4 exon 5. The gRNA was synthesized by Sigma to include additional hybridization sequences for tracrRNA. Embryos were microinjected at the single cell stage with 2nL of a solution containing gRNA, tracrRNA (cat # TRACRRNA05N; Sigma), S.pyogenes Cas9 (cat # M0386T; New England Biolabs) and phenol red. Each 2nL injection solution contained 130 pg of gRNA. Microinjected embryos were raised to adulthood. F0 chimeras were identified by genotyping the offspring of a wild-type outcross for loss of the targeted BamH1 site. Pooled genomic DNA was amplified using PCR primers 5ƍ- CCTATGGACATGGGAACAATCAA-3ƍ (SEQ ID NO:25) and 5ƍ- GGAACCCTATGCAGTTATCAAACTG-3ƍ (SEQ ID NO:26), yielding a 530 bp product spanning exon5 and intron 6 of brd4. BamHI digest of the WT PCR product results in restriction fragments of 333 bp and 197 bp, whereas mutation affecting the BamHI site results in an uncut 530bp product after digest. Offspring of F0 chimeras crossed to WT zebrafish were grown to adulthood and fin clip DNA obtained for genotyping by PCR and BamHI digest. This identified F1 founders, whose DNA was then sequenced to characterize mutations. Overall, we identified 5 unique F1 founders with different mutations (including deletions of 8 or 11 bp, insertion of 5bp and combined Attorney Docket No.: 49045-0085WO1 / 05762 deletion / insertions of 1 / 45 and 5 / 5 bp). We used the 11 bp deletion allele Pt435 for further studies, as it abolished Brd4 expression by western blot. The allele was outcrossed to WT zebrafish for 4 generations before being in-crossed, or crossed to the Tau zebrafish, for analysis. Survival studies Tg(elavl3:gal4-vp16); Tg(UAS:hsa.MAPT-2A-nls-mCherry) ‘Tau’ zebrafish, were identified by mCherry expression at 3dpf. Siblings not expressing mCherry, unrelated WT zebrafish, and Tg(elavl3:gal4-vp16); Tg(UAS:2A-nls-mCherry) ‘Ctrl’ zebrafish were used as controls for comparison. Zebrafish were maintained at identical initial density under standard conditions for zebrafish care after 5 days postfertilization (dpf), including fresh circulating water and feeding three times daily. Different experimental groups were maintained in separate tanks and the number of surviving zebrafish in each tank was counted daily until 15dpf. Cell death assays For acridine orange labeling, 2 – 7 dpf Tau and Ctrl zebrafish were immersed in acridine orange (cat # A6014, Sigma, St. Louis, MO) 5^g / mL in E3 embryo water in darkness for 30min. After three washes in E3, zebrafish were embedded individually in 1.5% agarose and imaged using an Olympus CKX41 inverted epifluorescence microscope. The number of fluorescently labeled cells in each spinal cord was counted manually. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Caspase 3 labeling, 2 – 7 dpf Tau and Ctrl zebrafish were fixed in 4% paraformaldehyde, washed in PBS, and cryoprotected in 30% sucrose. 12^m thick sections were collected in a sagittal plane using a cryostat. TUNEL labeling was carried out using a kit (TUNEL Andy Fluor 647 Apoptosis Kit, cat # A052, ABP Biosciences, Beltsville, MD), following the manufacturer’s protocol exactly. Cleaved (active) Caspase 3 was detected by immunohistochemistry as described below. Brain sections were imaged by widefield transmitted light or epifluorescence microscopy using an Olympus BX51 microscope. Labeled cells were quantified and normalized to tissue section area using ImageJ71, as shown herein. Attorney Docket No.: 49045-0085WO1 / 05762 Immunohistology Zebrafish were fixed and sectioned as above. Sections were mounted on glass slides, washed with phosphate-buffered saline + 0.3% Triton X-100 (PBST) 3 times for 5 minutes each, incubated for 60 minutes in blocking buffer (1% bovine serum albumin in PBST), then incubated with primary antibody (see below) at 4°C for 16 hours. After washing with PBST, sections were incubated with secondary antibody for 60 minutes at room temperature. For chromogenic labeling, biotinylated secondary antibodies (1:200 in PBS; cat # BA9200; Vector Laboratories, Burlingame, CA) were used, followed by incubation with HRP-avidin-biotin complexes (Vectastain, Vector Laboratories, Burlingame, CA), chromogenic histochemical reaction with 3,3'Diaminobenzidine substrate (Vector Laboratories), and counterstaining in Mayer’s hematoxylin (Sigma). For immunofluorescence, fluorophore-conjugated secondary antibodies – goat anti-mouse IgG Alexa Fluor 488, goat anti-rabbit IgG Alexa Fluor 488, donkey anti-goat IgG Alexa Fluor 555 (Invitrogen) – were diluted 1:1000 in PBS with DAPI (final concentration 200ng / mL in PBS; cat # 10236276001; Roche) as a nuclear counter-label. Primary antibodies and dilutions for immunohistology were as follows. Figure 6G: purified rabbit anti-active Caspase 3 (1:200; cat # 559565, BD Biosciences, San Jose, CA). Figure 6J: mouse anti-zebrafish microglial marker clone 7.4.C472(1:25; DSHB, University of Iowa). Figures 7E, 7F: mouse anti-human phospho(S202 / T205)-Tau (AT8; 1:500, Cat# MN1020, Thermo Fisher), mouse anti-human Tau IgG1 (MC1; 1:100; obtained from Dr. Peter Davies, Albert Einstein Medical College), mouse anti-human Tau IgM (Alz50; 1:100; obtained from Dr. Peter Davies, Albert Einstein Medical College), mouse anti-human phospho(T231)-Tau (AT180; 1:250; Cat# MN1040, Thermo Fisher), mouse anti-human phospho(T181)-Tau (AT270; 1:500; Cat# MN1050, Thermo Fisher), mouse anti-human phospho(S396 / S404)-Tau IgG1 (PHF1; 1:1000; obtained from Dr. Peter Davies, Albert Einstein College of Medicine), rabbit anti-human phospho(S422)- Tau (1:500; Cat# 44-764G, Thermo Fisher). Figure 7H: mouse anti-human truncated Tau (TauC3; 1:100; Cat# AHB0061, Invitrogen). Figure 11J: Rabbit anti-human Brd4 (1:200; Cat# ab128874, Abcam), goat anti-human IbaI (1:500; Cat# ab5076, Abcam). Whole mount immunofluorescence Zebrafish were dissected, then lysed and extracted with RIPA buffer (150mM NaCl, 10mM Tris-HCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% Sodium Attorney Docket No.: 49045-0085WO1 / 05762 deoxycholate) or DIGE buffer (7M Urea, 2M Thiourea, 30mM Tris-HCl, 4% CHAPS). For dephosphorylation assays in Figure 7B, samples were extracted with high salt buffer and proteins precipitated with saturated ammonia sulfate. The pellet was resuspended in 50mM Tris-HCl (pH8.0), 1mM MgCl2 and incubated with calf intestinal alkaline phosphatase (CIP; cat # M0525; New England Biolabs) or ^-protein phosphatase (^PP; cat # P0753; New England Biolabs) at 37°C for 60 minutes before electrophoresis. 60^g crude protein extract from each experiment group was loaded in each well of a 12% SDS-PAGE gel. After electrophoretic separation, proteins were transferred to nitrocellulose membrane (LI-COR), which was blocked using Odyssey blocking buffer (cat # 927-40000; LI-COR) for 60 minutes at room temperature followed by incubation with primary antibody (see below) at 4°C for 16 hours. After 3 washes of 10 minutes each in PBST, blots were incubated with secondary antibodies IRDye 800CW goat anti- Mouse IgG (1:10000; cat # 926-32210, LI-COR) and IRDye 680RD goat anti-Rabbit IgG (1:10000; cat #926-68071, LI-COR) in LI-COR blocking buffer for 60 minutes at room temperature in the dark. After 3 further washes in PBST, blots were scanned using a LiCOR Odyssey near infrared scanner and immunoreactive bands quantified using LI- COR Empiria software. Primary antibodies and dilutions for western blots were as follows. Figure 6G inset: rabbit anti-Caspase 3 (1:500; Cat# ab13847, Abcam). Figure 6H: mouse anti- Tyrosine Hydroxylase (1:1000; cat # MAB318, Millipore), rabbit anti-GAD65 / 67 (1:500; cat # AB1511, Millipore), rabbit anti-Synaptophysin (1:500; cat # ab32594, Abcam), rabbit anti-PSD95 (1:500; cat # ab18258, Abcam). Figures 7A, 7B, 7C, 7G, 7J, 10D: mouse anti-Tau[210-241] (Tau5; 1:1000; cat# AHB0042, Thermo Fisher), rabbit anti- human Tau[243-441] (1:5000; Cat# A0024, Dako), mouse anti-human phospho(S202 / T205)-Tau (AT8; 1:500, Cat# MN1020, Thermo Fisher), mouse anti- human phospho(T231)-Tau (AT180; 1:500; Cat# MN1040, Thermo Fisher), mouse anti- human phospho(T181)-Tau (AT270; 1:2000; Cat# MN1050, Thermo Fisher), mouse antihuman phospho(T212 / S214)-Tau (AT100; 1:250; Cat# MN1060, Thermo Fisher), mouse anti-human phospho(S396 / S404)-Tau IgG1 (1:1000; obtained from Dr. Peter Davies, Albert Einstein College of Medicine), rabbit anti-human phospho(S422)-Tau (1:1000; Cat# 44-764G, Thermo Fisher). Figure 11B: rabbit anti-Brd4 antibody (1:500; obtained from Dr. Igor Dawid, NIH)44. Rabbit anti-Actin (1:2000; cat # A2066, Sigma) was used to confirm equal protein loading on blots. Attorney Docket No.: 49045-0085WO1 / 05762 Analysis of motor activity Measurement of larval zebrafish motor activity (Figure 8A – 8F, Figure 4, Figure 10A – 10C, Figure 11C – 11D) was carried out using the open-source MATLAB applications LSRtrack and LSRanalyze33exactly as described in our prior work68,70and in our detailed published protocol31. Briefly, 5dpf larvae were transferred to 96 well plates with black well surrounds and optical glass bottoms (Corning 96-well Special Optics Microplate; cat # CLS3720; Sigma) using a large-bore Pasteur pipette with a flame- polished aperture, then acclimatized to the recording chamber for 30 min at 28.5ÛC in white light (200 lx, color temperature 3500K). Motor responses were recorded under continuous ambient illumination for 60 minutes, and then the visual motor response was elicited during 3 cycles each of (10 minutes dark + 10 minutes light). Recordings of trans-illuminated zebrafish were made through the glass bottom of the 96-well plate, with an infrared light source (#BL812-880, Spectrum Illumination, Montague, MI) positioned above and a USB 3.0 camera (#FL3-U3-13Y3M-C, Point Gray Research, Richmond, BC, Canada) with a 50mm lens and IRpass filter positioned 1.2m below. Video recordings were analyzed offline. Analysis of kinematics Analysis of truncal swimming kinematics was carried out using our open-source MATLAB application HiSpeedTracking, exactly as reported in our previous work34. Briefly, 5dpf zebrafish were transferred to 15 mm diameter wells cut into a 0.3% agarose- filled plate and allowed to acclimatize to the recording chamber at 28.5°C for 30 min. ‘O’-bend responses were elicited over 40 stimulus cycles of abrupt transition between bright white light ambient illumination (1100 lx, 4900 K) and dark (<1 lx). Video was captured from below the plate using infrared illumination as described above but using a high-speed camera (Integrated Design Tools, Pasadena, CA; model #NX8- S2) with a macro lens (Rokinon 100 mm f2.8; B&H, New York) and infrared-pass filter (R72, 720 nm; B&H). Video segments of 1 s duration at 1000 frames / s were captured at each light- dark transition, synchronized to the stimulus using a USB relay. Video recordings were analyzed offline. Analysis of optokinetic reflex responses Attorney Docket No.: 49045-0085WO1 / 05762 Analysis of optokinetic reflexes was carried out using our open-source MATLAB applications OKRtrack and OKRanalyze36, exactly as reported in our previous work35. Briefly, 5 dpf zebrafish larvae were immobilized in 3.5% methylcellulose on a transparent platform suspended within a cylindrical backprojection screen. Larvae were illuminated by an infrared light source from below (880 nm; #BL34-880, Spectrum, Montague, MI) and responses captured from above at 30 frames / s, using a zoom microscope (#S8 APO, Leica Microsystems, Wetzlar, Germany) with an IR-sensitive camera (#FL3-U3-13Y3M-C, Point Grey Research, Richmond, BC, Canada). A sinusoidal grating pattern (15° / cycle, 99% contrast, and sinusoidally-transformed to appear linearly spaced from within the cylinder) was projected onto the screen so that the pattern filled the left visual field of the zebrafish, and the stimulus was animated so it appeared to move at 15° / s (=1 cycle / s). Reflex gain was analyzed over 60s of stimuli alternating every 6s between nasotemporal (NT) and temporonasal (NT) directions. Optokinetic nystagmus was then provoked by uninterrupted NT or TN stimuli for 60s in each direction to evaluate saccades and movement range. Video recordings were analyzed offline. Chemical exposure and phenotypic screening An epigenetics screening library (Cayman Chemical, Ann Arbor, MI; cat # 11076; batch # 0468238) provided 140 chemical inhibitors dissolved in DMSO at 10mM. Zebrafish were exposed to chemicals in 6-well plates, starting at 2 dpf, with water change and fresh chemical at 3dpf and 4dpf, and phenotypic readouts at 5dpf. The first step of the screen involved testing each chemical in WT zebrafish at 50^M in E3 embryo buffer (yielding a maximum DMSO concentration of 0.5%, which is within the limit of tolerance for zebrafish larvae73,74). Any chemical causing overt toxicity (death, morphological abnormalities by light microscopy, or obvious neurological problems) was tested at progressively lower concentrations until the maximum tolerated concentration (MTC) of each chemical had been established. Tau zebrafish were then exposed to the MTC of each chemical from 2 – 5 dpf as above, in groups of 12 zebrafish per well of a 6- well plate. At 5dpf, chemicals were washed off and zebrafish transferred in fresh E3 buffer to 96-well plates for motor activity measurements as described above. Groups of 12 Tau and non-expressing sibling zebrafish were grown in parallel under identical conditions but without chemical exposure, to provide controls for the motor activity Attorney Docket No.: 49045-0085WO1 / 05762 assays. All experiments were completed without reference to the identity of the chemicals; the library key was only unblinded after analysis of screening data was completed. Data analysis workflows were automated using custom MATLAB scripts to ensure assay QC criteria were met, and to quantify chemical rescue of the Tau phenotype across the VMR, while averaging responses of individual zebrafish to multiple light cycles to minimize variability. (+)JQ1 and (í)JQ1 were repurchased from Sigma (cat #s SML1524 and SML1525) for the experiments shown in Figure 9F and Figure 10. References for this Example: 1. Steele, J.C., Richardson, J.C. & Olszewski, J. Progressive Supranuclear Palsy. 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Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer's disease. Proc Natl Acad Sci U S A 100, 10032-7 (2003). 29. Wittmann, C.W. et al. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293, 711-4 (2001). 30. de Calignon, A. et al. Caspase activation precedes and leads to tangles. Nature 464, 1201-1204 (2010). 31. Zhou, Y., Cattley, R.T., Cario, C.L., Bai, Q. & Burton, E.A. Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications. Nat Protoc 9, 1533-48 (2014). 32. Farrell, T.C. et al. Evaluation of spontaneous propulsive movement as a screening tool to detect rescue of Parkinsonism phenotypes in zebrafish models. Neurobiol Dis 44, 9-18 (2011). 33. Cario, C.L., Farrell, T.C., Milanese, C. & Burton, E.A. Automated measurement of zebrafish larval movement. J Physiol 589, 3703-8 (2011). 34. Hossainian, D. et al. Quantification of functional recovery in a larval zebrafish model of spinal cord injury. J Neurosci Res 100, 2044-2054 (2022). 35. Shao, E., Scheetz, S.D., Xie, W. & Burton, E.A. Modulation of the zebrafish optokinetic reflex by pharmacologic agents targeting GABAA receptors. Neurosci Lett 671, 33-37 (2018). 36. Scheetz, S.D. et al. An open-source method to analyze optokinetic reflex responses in larval zebrafish. J Neurosci Methods 293, 329-337 (2018). Attorney Docket No.: 49045-0085WO1 / 05762 37. Garbutt, S. et al. Abnormalities of optokinetic nystagmus in progressive supranuclear palsy. Journal of Neurology, Neurosurgery & Psychiatry 75, 1386-1394 (2004). 38. Long, K., Kostman, S.J., Fernandez, C., Burnett, J.C. & Huryn, D.M. Do Zebrafish Obey Lipinski Rules? ACS Med Chem Lett 10, 1002-1006 (2019). 39. Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712-716 (2016). 40. Briel, N., Pratsch, K., Roeber, S., Arzberger, T. & Herms, J. Contribution of the astrocytic tau pathology to synapse loss in progressive supranuclear palsy and corticobasal degeneration. Brain Pathology 31, e12914 (2021). 41. Bigio, E.H. et al. Cortical Synapse Loss in progressive Supranuclear palsy. Journal of Neuropathology & Experimental Neurology 60, 403-410 (2001). 42. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067-73 (2010). 43. Huang, B., Yang, X.D., Zhou, M.M., Ozato, K. & Chen, L.F. Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA. Mol Cell Biol 29, 1375-87 (2009). 44. Toyama, R., Rebbert, M.L., Dey, A., Ozato, K. & Dawid, I.B. Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis. Dev Dyn 237, 1636- 44 (2008). 45. Sjöstedt, E. et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. Science 367, eaay5947 (2020). 46. Rossaert, E. et al. Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model. Acta Neuropathologica Communications 7, 107 (2019). 47. Merelli, A. et al. Understanding the Role of Hypoxia Inducible Factor During Neurodegeneration for New Therapeutics Opportunities. Curr Neuropharmacol 16, 1484-1498 (2018). 48. Weber, A. et al. Epigenome-wide DNA methylation profiling in Progressive Supranuclear Palsy reveals major changes at DLX1. Nature Communications 9, 2929 (2018). 49. Shi, Q. et al. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP / PS1 mice. Sci Transl Med 9(2017). 50. Wu, T. et al. Complement C3 Is Activated in Human AD Brain and Is Required for Attorney Docket No.: 49045-0085WO1 / 05762 Neurodegeneration in Mouse Models of Amyloidosis and Tauopathy. Cell Rep 28, 2111- 2123.e6 (2019). 51. Dejanovic, B. et al. Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies. Neuron 100, 1322-1336 e7 (2018). 52. Litvinchuk, A. et al. Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer's Disease. Neuron 100, 1337-1353 e5 (2018). 53. Zhou, J. et al. The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration. Science Translational Medicine 15, eadf0141 (2023). 54. Wasiak, S. et al. Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208). Journal of Cardiovascular Translational Research 10, 337-347 (2017). 55. Ising, C. et al. NLRP3 inflammasome activation drives tau pathology. Nature 575, 669-673 (2019). 56. Yoshiyama, Y. et al. Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53, 337-51 (2007). 57. Zhang, S. & Cui, P. Complement system in zebrafish. Dev Comp Immunol 46, 3-10 (2014). 58. Mazzolini, J. et al. Gene expression profiling reveals a conserved microglia signature in larval zebrafish. Glia 68, 298-315 (2020). 59. Peri, F. & Nusslein-Volhard, C. Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133, 916-27 (2008). 60. Cochran, A.G., Conery, A.R. & Sims, R.J. Bromodomains: a new target class for drug development. Nature Reviews Drug Discovery 18, 609-628 (2019). 61. Sun, Y. et al. Safety and Efficacy of Bromodomain and Extra-Terminal Inhibitors for the Treatment of Hematological Malignancies and Solid Tumors: A Systematic Study of Clinical Trials. Front Pharmacol 11, 621093 (2020). 62. Gibbons, H.R. et al. Bromodomain inhibitor JQ1 reversibly blocks IFN-Ȗ production. Scientific Reports 9, 10280 (2019). Attorney Docket No.: 49045-0085WO1 / 05762 63. Magistri, M. et al. The BET-Bromodomain Inhibitor JQ1 Reduces Inflammation and Tau Phosphorylation at Ser396 in the Brain of the 3xTg Model of Alzheimer's Disease. Curr Alzheimer Res 13, 985-95 (2016). 64. Wang, H. et al. (+)-JQ1 attenuated LPS-induced microglial inflammation via MAPK / NFkappaB signaling. Cell Biosci 8, 60 (2018). 65. Malpetti, M. et al. Neuroinflammation and Tau Colocalize in vivo in Progressive Supranuclear Palsy. Ann Neurol 88, 1194-1204 (2020). 66. Holland, N. et al. Molecular pathology and synaptic loss in primary tauopathies: an 18F-AV-1451 and 11C-UCB-J PET study. Brain 145, 340-348 (2022). 67. Asakawa, K. et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci U S A 105, 1255-60 (2008). 68. Van Laar, V.S. et al. Į-Synuclein amplifies cytoplasmic peroxide flux and oxidative stress provoked by mitochondrial inhibitors in CNS dopaminergic neurons in vivo. Redox Biol 37, 101695 (2020). 69. Dukes, A.A. et al. Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP(+) exposure. Neurobiol Dis 95, 238-49 (2016). 70. Xie, W. et al. Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity. eLife 9, e51845 (2020). 71. Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671-675 (2012). 72. Bai, Q., Parris, R.S. & Burton, E.A. Different mechanisms regulate expression of zebrafish myelin protein zero (P0) in myelinating oligodendrocytes and its induction following axonal injury. J Biol Chem 289, 24114-28 (2014). 73. Christou, M., Kavaliauskis, A., Ropstad, E. & Fraser, T.W.K. DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls. Science of The Total Environment 709, 134490 (2020). 74. Hoyberghs, J. et al. DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay. Frontiers in Toxicology 3(2021). Example 2 – Use of midostaurin to treat progressive supranuclear palsy Attorney Docket No.: 49045-0085WO1 / 05762 A human identified as having a tauopathy is administered (e.g., orally administered) from about 50 mg to about 100 mg of midostaurin twice a day for about 3 to about 4 weeks. In some cases, such a cycle of administration of midostaurin can be repeated two or more times. The administered midostaurin can improve one or more neurological functions within the human. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 49045-0085WO1 / 05762 WHAT IS CLAIMED IS:

1. A method of treatment of tauopathy, comprising: administering a pharmaceutical preparation comprising a pharmaceutically effective amount of a compound selected from the group of staurosporine, midostaurin, and biologically active analogs thereof.

2. The method of treatment of claim 1, wherein the compound is midostaurin.

3. The method of treatment of claim 1, wherein the tauopathy is progressive supranuclear palsy.

4. A method for treating a mammal having a tauopathy, wherein said method comprises administering, to said mammal, a composition comprising a pharmaceutically effective amount of a compound selected from the group consisting of staurosporine, midostaurin, and biologically active analogs thereof.

5. The method of claim 4, wherein said mammal is a human.

6. The method of claim 4, wherein said compound is midostaurin.

7. The method of claim 4, wherein said tauopathy is progressive supranuclear palsy.

8. The method of claim 4, wherein said method comprises identifying said mammal as having said tauopathy prior to said administering step.

9. A method of screening for activity in treatment of tauopathy, comprising: selecting one or more agents to screen; for each one of the one or more agents, exposing one or more transgenic zebrafish expressing human Tau to the one of the one or more agents, and comparing neurological function of the transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents to neurological function of zebrafish expressing human Tau which were not exposed to the one of the one or more agents.Attorney Docket No.: 49045-0085WO1 / 05762 10. The method of claim 9, comprising: for each one of the one or more agents, exposing a group of transgenic zebrafish expressing human Tau to the one of the one or more agents, and comparing neurological function of the group of transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents to neurological function of the group of zebrafish expressing Tau which were not exposed to the one of the one or more agents.

11. The method of any one of claims 9 and 10, wherein comparing neurological function of the transgenic zebrafish expressing human Tau and exposed to the one of the one or more agent to the neurological function of the transgenic zebrafish expressing human Tau which were not exposed to the one of the one or more agent comprises comparison thereof to neurological function of one or more control zebrafish not expressing human Tau.

12. The method of any one of claims 9-11, wherein the transgenic zebrafish expressing human Tau express wildtype or non-mutant human Tau.

13. The method of claim 12, wherein the transgenic zebrafish expressing human Tau express 4R-Tau.

14. The method of claim 12, wherein the transgenic zebrafish expressing human Tau express 0N / 4R-Tau or human P301L 0N / 4R-Tau.

15. The method of any one of claims 10-14, wherein one or more neurological functions of a plurality of transgenic zebrafish expressing human Tau within the group of transgenic zebrafish expressing human Tau are compared to one or more neurological functions of a plurality of transgenic zebrafish expressing human Tau which were not exposed to the one of the one or more agents.

16. The method of claim 15, further comprising quantifying the one or more neurological functions and comparing a mean of each of the one or more neurologicalAttorney Docket No.: 49045-0085WO1 / 05762 functions of the plurality of transgenic zebrafish expressing human Tau and exposed to the one of the one or more agents with a corresponding mean of the one or more neurological functions of the plurality of the zebrafish expressing human Tau which were not exposed to the one of the one or more agents.

17. The method of claim 15, wherein the one or more neurological functions are selected from swimming motion and eye motion.

18. The method of any one of claims 9-17, wherein the one or more agents to screen are selected from at least one of Brd4 inhibitors, histone deacetylases inhibitors, DNA methyltransferase inhibitors, or kinase inhibitors.

19. The method of any one of claims 9-17, wherein the one or more agents to screen are selected from at least one of known Brd4 inhibitors or kinase inhibitors.

20. The method of any one of claims 9-19, wherein each of the one or more neurological functions is quantified via software-based analysis of videographic data.

21. The method of any one of claims 9-20, wherein the transgenic zebrafish expressing human Tau comprise one or more transgene alleles comprising an encoded GAL4 driver and a UAS responder cassette comprising encoded human Tau, an encoded imaging reporter, and a self-cleaving peptide separating the encoded human Tau and the encoded imaging reporter.

22. The method of any one of claims 9-20, wherein the transgenic zebrafish expressing human Tau comprise one or more transgene alleles comprising an encoded GAL4 driver and UAS responder cassette comprising encoded wild type human 0N / 4R- Tau, an encoded imaging reporter, and a self-cleaving peptide separating the encoded wild type human 0N / 4R-Tau and the encoded imaging reporter.

23. The method of any one of claims 21-22, wherein the encoded imaging reporter is a fluorescent protein.Attorney Docket No.: 49045-0085WO1 / 05762 24. The method of any one of claims 21-23, wherein the encoded imaging reporter is mCherry.

25. The method of any one of claims 21-24, wherein a targeting signal is associated with the encoded imaging reporter so that the human Tau and the imaging reporter are expressed independently in separate tissue compartments.

26. The method of claim 25, wherein the targeting signal is a nuclear localization signal (nls).

27. The method of any one of claims 9-26, wherein the one or more agents are one or more chemical compounds.

28. The method of any one of claims 9-27, wherein the method is a high throughput screening.

29. The method of any one of claims 9-28, wherein the tauopathy is progressive supranuclear palsy.

30. A method for identifying an agent for treating a tauopathy within a mammal, wherein said method comprises: (a) exposing one or more transgenic zebrafish expressing a human Tau to a test agent; and (b) determining the presence of an increased level of a neurological function of said one or more transgenic zebrafish expressing said human Tau and exposed to said test agent as compared to the level of said neurological function of one or more zebrafish expressing said human Tau that were not exposed to said test agent, thereby identifying said test agent as being said agent for treating said tauopathy within said mammal.

31. The method of claim 30, wherein said transgenic zebrafish expressing said human Tau express wildtype or non-mutant human Tau.Attorney Docket No.: 49045-0085WO1 / 05762 32. The method of claim 31, wherein said transgenic zebrafish expressing said human Tau express 4R-Tau.

33. The method of claim 31, wherein said transgenic zebrafish expressing said human Tau express 0N / 4R-Tau or human P301L 0N / 4R-Tau.

34. The method of any one of claims 30-33, wherein said neurological function is selected from the group consisting of swimming motion and eye motion.

35. The method of any one of claims 30-34, wherein said test agent is selected from the group consisting of Brd4 inhibitors, histone deacetylases inhibitors, DNA methyltransferase inhibitors, and kinase inhibitors.

36. The method of any one of claims 30-35, wherein said neurological function is quantified via software-based analysis of videographic data.

37. The method of any one of claims 30-36, wherein said transgenic zebrafish expressing said human Tau comprise one or more transgene alleles comprising an encoded GAL4 driver and a UAS responder cassette comprising encoded human Tau, an encoded imaging reporter, and a self-cleaving peptide separating said encoded human Tau and said encoded imaging reporter.

38. The method of any one of claims 30-36, wherein said transgenic zebrafish expressing said human Tau comprise one or more transgene alleles comprising an encoded GAL4 driver and UAS responder cassette comprising encoded wild type human 0N / 4R-Tau, an encoded imaging reporter, and a self-cleaving peptide separating said encoded wild type human 0N / 4R-Tau and said encoded imaging reporter.

39. The method of any one of claims 37-38, wherein said encoded imaging reporter is a fluorescent protein.

40. The method of any one of claims 37-39, wherein said encoded imaging reporter is mCherry.Attorney Docket No.: 49045-0085WO1 / 05762 41. The method of any one of claims 37-40, wherein a targeting signal is associated with said encoded imaging reporter so that said human Tau and said imaging reporter are expressed independently in separate tissue compartments.

42. The method of claim 41, wherein said targeting signal is a nuclear localization signal (nls).

43. The method of any one of claims 30-42, wherein said tauopathy is progressive supranuclear palsy.

44. The method of any one of claims 30-43, wherein said mammal is a human.

45. A method for treating a human having a tauopathy, wherein said method comprises administering, to said human, a composition comprising from about 50 mg to about 100 mg of midostaurin.

46. The method of claim 45, wherein said composition is administered from 1 to about four times a day.

47. The method of claim 45, wherein said composition is administered twice a day.

48. The method of any one of claims 45-47, wherein said tauopathy is progressive supranuclear palsy.

49. A method for treating a human having progressive supranuclear palsy, wherein said method comprises administering, to said human, a composition comprising from about 50 mg to about 100 mg of midostaurin.

50. The method of claim 49, wherein said composition is administered from 1 to about 4 times a day.

51. The method of claim 49, wherein said composition is administered 2 times a day.

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