DOI: 10.5061/dryad.fqz612k0k
title: Protein assembly modulation: A new approach to Amyotrophic Lateral Sclerosis (ALS) therapeutics

description: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a
 complex, multifactorial pathophysiology, most commonly manifest as loss of
 motor neurons. We introduce a new mechanism of ALS pathogenesis via a
 novel drug-like small molecule series that targets a subset of protein
 disulfide isomerase (PDI) within a previously unappreciated transient and
 energy-dependent multi-protein complex. This drug was found to have
 activity in cellular models for both familial and sporadic ALS, as well as
 in transgenic worms, flies, and mice bearing a diversity of human genes
 with ALS-associated mutations. The hit compound was initially identified
 as a modulator of human immunodeficiency virus (HIV) capsid assembly in
 cell-free protein synthesis and assembly (CFPSA) systems, with
 demonstrated antiviral activity in cell culture. Its advancement for
 ALS-therapeutics, and subsequent separation of activity against HIV and
 ALS into separate chemical subseries through
 structure-activity-relationship optimization, may provide insights into
 the molecular mechanisms governing pathophysiology of disordered
 homeostasis relevant to ALS.
description: <strong><em>In vitro
 studies</em></strong> <strong>Stress
 granule aggregation in SH-SY5Y cells overexpressing
 TDP-43</strong> A TDP-43 cellular model was
 successfully generated by establishing stable cell-lines over-expressing
 wild-type TDP-43 and M337V mutant TDP-43 and showing the display of stress
 granules, following arsenite treatment. SH-SY5Y tet-on TDP-43 partial
 3`-UTR (wt or M337V), 50.000 cells (DMEM/F12 + 1 µg/mL doxycycline)
 incubated on cover slips overnight. Cells were treated sodium arsenite at
 a final concentration of 250 µM and incubated for 90 min. Cells were
 washed with 500 µL PBS.  Cells were fixed with 4% PFA in PBS pH 7.4 for 15
 min at RT.  Cells were washed with 500 µL PBS. Permeabilize/ block cells
 with 5% milk powder, 1% BSA and 0.5 % saponin in PBS for 45 min @ RT. Add
 respective {TDP-43 C-terminal domain Antibody (1:1000; Purchased from
 Proteintech), HuR (1:500; Purchased from Santa Cruz} in 1% PBS and 0.5%
 saponine in PBS and incubated overnight at  4°C.  Cells were washed 3 x
 with 500 µL PBS and incubated with secondary antibody at a dilution of
 1:1000 AlexaFluor a-rabbit 594 (highly cross-adsorbed) (Thermo Fisher) or
 AlexaFluor a-mouse 488 (highly cross-adsorbed) (Thermo Fisher).  Cells
 were washed 3x with 500 µL PBS, then cells were washed with 500 µL dH2O. 
 ProLong Gold with DAPI embedding medium was used to fix cells on a glass
 slide. Image collection was done on a Zeiss AxioImager 2 equipped with an
 Apotome using the following filtersets. For the automated image analysis,
 each raw grayscale channel image was saved and analyzed independently. 
 Each image set was calibrated using the TDP-43 induced, sodium arsenite
 treated condition as a reference for exposure time of the different
 channels.                                           
 <strong>High-Content Imaging of Endogenous TDP-43 Stress
 Granule (SG):</strong> On day 1, seed 20,000
 patient derived fibroblasts per well in a 24 well glass bottom plate or
 6000 cells per well in a 96 well plate. On day 2, sonicate compounds for
 10 mins at 37oC before use. Add compounds at the desired final
 concentration in fresh media to the respective wells. Add equivalent
 amount of DMSO (LC-MS grade) to control wells. On Day 3, add sodium
 arsenite treatment- Add sodium arsenite at a final concentration of 500uM.
 Incubate at 37oC for 60 mins. Wash 1X with PBS and fix cells with 4% para
 formaldehyde (in PBS, prepared freshly, methanol free) for 15 mins at room
 temperature. Wash 3X with PBS. Permeabilization and
 Blocking- Add 0.1% Triton-X for 10 mins for permeabilization followed by 1
 hour of blocking in 1% BSA. Immunostaining- Add the following primary
 antibodies in 1% BSA (in PBS) and incubate it overnight at 4oC. Rabbit
 polyclonal TDP-43 C-terminal antibody (Proteintech 12892-1-AP)- 1:450;
 mouse monoclonal HuR antibody (Santa Cruz sc-5261)- 1:500. On day 4, wash
 3X with PBST (PBS + 0.1% Tween). The following secondary antibodies from
 Thermofisher Scientific (1:500) in 1% BSA (in PBS) and keep it in dark for
 1-2 hours at room temperature. Alexa 594 anti-rabbit
 (highly cross-adsorbed); Alexa 488 anti-mouse (highly cross-adsorbed);
 Wash 3X with PBST in dark. Add DAPI in PBS for nuclear staining. Imaging
 and Image analysis is done as explained below. In brief, the
 immuno-stained cells were imaged with Nikon Ti inverted fluorescence
 microscope having CSU-22 spinning disk confocal and EMCCD camera. Plan Apo
 objectives and NIS-Elements AR software were used for image acquisition.
 At least 30-50 images per well is taken.      
 <strong>Nucleocytoplasmic assay in FTD
 PDFs:                                                                  
 </strong> Skin-derived fibroblasts cells from a
 sporadic Frontotemporal Degeneration (FTD) and ALS affected individual
 acquired from the National Institute of Neurological Disorders and Stroke
 were grow in HyClone DMEM High Glucose (GE Healthcare Life Sciences)
 supplemented with 15% FBS and 1% NEAA (Non-Essential Amino Acids), at 37ºC
 in an humidified atmosphere of 5% CO2. On Day 1, seed 600 cells per well
 in a 96 well glass bottom plate or 1200 cells per well in a 24 well glass
 bottom plate. Incubate for 4 days, at 37ºC in an humidified atmosphere of
 5% CO2. On day 5,                sonicate compounds for 10 mins at 37oC
 before use. Add compounds at the desired final concentration in fresh
 media to the respective wells. Add equivalent amount of DMSO (LC-MS grade)
 to control wells. Incubate for 4 days, at 37ºC in a humidified atmosphere
 of 5% CO2. On day 9, wash 2X with PBS. To fix add 4% para formaldehyde (in
 PBS, prepared freshly, methanol free) for 15 mins at room temperature.
 Wash 3X with PBS. Blocking and permeabilization- Add 1% BSA + 1% saponin
 (prepared in PBS) for 1 hour. Immunostaining- Add the
 following primary antibodies in 1% BSA (in PBS) and incubate it overnight
 at 4oC. Rabbit polyclonal TDP-43 C-terminal antibody (Proteintech
 12892-1-AP)- 1:350; mouse monoclonal HuR antibody (Santa Cruz sc-5261)-
 1:500. On day 10, wash 3X with PBST (PBS + 0.1% Tween). Add the following
 secondary antibodies from Thermofisher Scientific (1:500) in 1% BSA (in
 PBS) and keep it in dark for 1-2 hours at room temperature. Alexa 594
 anti-rabbit (highly cross-adsorbed). Alexa 488 anti-mouse (highly
 cross-adsorbed). Wash 3X with PBST in dark. Add DAPI in PBS for nuclear
 staining. The immuno-stained cells are imaged with Nikon Ti inverted
 fluorescence microscope having CSU-22 spinning disk confocal and EMCCD
 camera. Plan Apo 20x/0.75 objective and NIS-Elements AR software were used
 for image acquisition. At least 15 images per well are taken. The exposure
 times for TDP-43 and HuR must remain constant across one experiment. Each
 image acquired (in .nd format) is exported into three individual channel
 images (for DAPI, TDP-43, HuR) in .tiff format. The images are analyzed by
 the open source image analysis software Cell Profiller. The DAPI image is
 used to count the total number of cells. The TDP-43 and HuR images are
 used to count the number of cells containing TDP-43 and/or HuR nuclear
 staining.          <strong>Automated Image
 Analysis and Machine Learning
 Tools:                                                                                              </strong> The immuno-stained cells were imaged with Nikon Ti inverted fluorescence microscope having CSU-22 spinning disk confocal and EMCCD camera. Plan Apo objectives and NIS-Elements AR software were used for image acquisition. At least 30-50 images per well is taken. The exposure times for TDP-43 and HuR must remain constant across one experiment. Each image acquired (in .nd format) is exported into three individual channel images (for DAPI, TDP-43, HuR) in .tiff format. The images were analyzed by the open source image analysis software CellProfiler 2.1.1 (offered by Broad Institute of Harvard and MIT- www.cellprofiler.org). This software contains various modules which can be used to analyze images in different ways. A Cell Profiler pipeline from a few of these modules was established to analyze our images in order to quantify the number of cytoplasmic TDP-43 positive HuR stress granules. The outline of the Cell Profiler pipeline: The three channel images are loaded and named DAPI, TDP-43 or HuR. Identify primary objects- The DAPI image is used to identify the nucleus as an object. Identify secondary objects- The nucleus is used to identify the cell boundary in the TDP-43 image by signal propagation. Identify tertiary objects- Based on the nucleus and the cell boundary, cytoplasm is identified as an object. Mask images- Using the cytoplasm object, the TDP-43 and HuR images are masked such that only cytoplasmic signal will remain. Enhance features: Enhance the signal from TDP-43 and HuR aggregates in cytoplasm for efficient identification of the aggregates. Identify primary objects- The TDP-43 aggregates in the cytoplasm were identified from the TDP-43 image and HuR aggregates were identified from the HuR image. Relate objects- This module enables the calculation of the number of TDP-43 aggregates which has HuR and vice versa. Export to spreadsheet- This module exports all data into Excel sheets. The final data has to be curated from the Excel sheets generated by Cell Profiler. The outlines for the different objects (nucleus, cytoplasm, aggregates) must be saved to cross-check the proper identification of objects once the analysis is done. At the beginning of analysis of an experiment, few images (from DMSO wells) must be used as training set for Cell Profiler. Based on the training set, the pipeline must be optimized with respect to intensity threshold, algorithm and size parameters for correct identification of primary and secondary objects (nucleus, cytoplasm, aggregates). It is extremely important to optimize this pipeline for every experiment. The pipeline must remain constant with respect to aggregate identification for the analysis of all the images from the same experiment. Finally, we calculate the number of TDP-43 positive HuR stress granules from the Excel sheets generated at the end of Cell Profiler.                                                                                                  <strong>                                                                   </strong> <strong>HIV infectious virus assay </strong> MT-2 cells were preseeded in 96-well plates in 100 ul of complete RPMI. Multiple concentrations of PAV-951 were serially diluted in DMSO then into an infection media prepared by diluting NL4-3 Rluc virus stock to 400 IU/100 ul with complete RPMI, which was transferred onto the MT-2 cells with a final MOI of 0.02 and final DMSO concentration of 1% in infected places. One well received DMSO only, instead of PAV-951, and one well received medium only for normalization and background collection. Cells were incubated at 37<sup>o</sup> C for 96 hours. 100ul of medium was removed and discarded and 10 ul of 15 uM EnduRen luciferase substrate was added to each well, followed by incubation for 1.5 hours at 37<sup>o</sup> C. Plates were read on a luminescence plate reader. Bioluminescence intensity was read on a Synergy H1 BioTek plate reader. Averages and standard deviation for viral titer observed under different treatment conditions were calculated in Microsoft Excel and graphed as the percent inhibition in PAV-951 treated cells compared to untreated cells. <strong>Drug Resin affinity chromatography</strong> Mouse brains from wildtype or SOD1 mutant animals were homogenized in cold phosphate buffered saline (PBS) (10mM sodium phosphate, 150 mM sodium chloride pH 7.4), then spun at 1,000 rpm for 10 minutes until pelleted. The PBS was decanted and the pellet resuspended in a low salt buffer (10mM HEPES pH 7.6, 10mM NaCl, 1mM MgAc with 0.35% Tritonx100) then centrifuged at 10,000 rpm for 10 minutes at 4<sup>o</sup>C. The post-mitochondrial supernatant was removed and adjusted to a concentration of approximately 10 mg/ml and equilibrated in a physiologic column buffer (50 mM Hepes ph 7.6, 100 mM KAc, 6 mM MgAc, 1 mM EDTA, 4mM TGA). In some conditions, the extract was supplemented with an energy cocktail (to a final concentration of 1mM rATP, 1mM rGTP, 1mM rCTP, 1mM rUTP, and 5 ug/mL creatine kinase). 30 ul or 230 ul of extract was then incubated for one hour at either 4<sup>o</sup> C or 22<sup>o</sup> C degrees on 30 ul of affigel resin coupled to THIQ compound or a 4% agarose matrix (control). The input material was collected and the resin was then washed with 3 ml column buffer. The resins were eluted for 2 hours then overnight at 22<sup>o</sup>C then 4<sup>o</sup>C in 100ul column buffer containing 100uM of the cognate compound. Eluates were run on western blot or sent for mass spectrometry for analysis. <strong>Chemical photocrosslinking</strong> Extract from mouse brain and PDFs grown in minimum essential media were prepared as above then adjusted to a protein concentration of approximately 3 mg/ml in column buffer containing 0.01% triton. 1% DMSO or 100uM PAV-073 was added to 6ul of extract, then 3uM of PAV-073 photocrosslinker or a negative control crosslinker (comprising of the biotin and diazirine moieties without compound) were added. The extract was incubated for 20 minutes then exposed to UV at 365nM wavelength for 10 minutes then left on ice for one hour. After crosslinking, samples were divided in two 20 ul aliquots and one set was denatured by adding 20 uL of column buffer 4ul of 10% SDS, 0.5 ul 1M DTT, and boiling for 5 minutes. Both native and denatured aliquots were then diluted in 800 ul column buffer containing 0.1% triton. 5 ul of magnetic streptavidin beads (Pierce) were added to all samples and mixed for one hour at room temperature to capture all biotinylated proteins and co-associated proteins.  Samples were placed on a magnetic rack to hold the beads in placed and washed three times with 800 ul of column buffer containing 0.1% triton. After washing, beads were resuspended in 80 ul of gel loading buffer containing SDS and analyzed by western blot or blot for affinity purified streptavidin. Samples were analyzed by western blot. <strong>Western blotting</strong> SDS/PAGE gels were transferred in Towbin buffer (25mM Tris, 192mM glycine, 20% w/v methanol) to polyvinylidene fluoride membrane, blocked in 1% bovine serum albumin (BSA) in PBS, incubated overnight at 4<sup>o</sup>C in a 1:1,000 dilution of 100ug/mL affinity-purified primary IGG to PDI in 1% BSA in PBS containing 0.1% Tween-20 (PBST). Membranes were then washed twice in PBST and incubated for two hours at room temperature in a 1:5000 dilution of secondary anti-rabbit or anti-mouse antibody coupled to alkaline phosphatase in PBST. Membranes were washed two more times in PBST then incubated in a developer solution prepared from 100 uL of 7.5 mg/mL 5-bromo-4-chloro-3-indolyl phosphate dissolved in 60% dimethyl formamide (DMF) in water and 100ul of 15 mg/ml nitro blue tetrazolium dissolved in 70% DMF in water, adjusted to 50mL with 0.1 Tris (pH 9.5) and 0.1 mM magnesium chloride. Membranes were scanned and the integrated density of protein band was measured on ImageJ. Averages and the standard deviation between repeated experiments were calculated and plotted on Microsoft Excel. <strong>Tandem mass spectrometry</strong> Samples were processed by SDS PAGE using a 10% Bis-tris NuPAGE gel with the 2-(N-morpholino)ethanesulfonic acid buffer system. The mobility region was excised and washed with 25 mM ammonium bicarbonate followed by 15mM acetonitrile. Samples were reduced with 10 mM dithoithreitol and 60<sup>o</sup> C followed by alkylation with 5o mM iodoacetamide at room temperature. Samples were then digested with trypsin (Promega) overnight (18 hours) at 37<sup>o</sup> C then quenched with formic acid and desalted using an Empore SD plate. Half of each digested sample was analyzed by LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75 uM analytical column at 350 nL/min packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in a data dependent mode, with the Oribtrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS respectively. The fifteen most abundant ions were selected for MS/MS. Data was searched using a local copy of Mascot (Matrix Science) with the following parameters: Enzyme: Trypsin/P; Database: SwissProt Human (conducted forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C) Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N/Q) Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. The data was analyzed by spectral count methods. <strong><em>In vivo studies</em></strong> <strong>Transgenic Human TDP-43 mutant <em>C. elegans</em>                                                                    </strong> MosSCI homologous-recombination transgenesis was used to create an unc- 47p::hTDP-43::unc-54utr or unc-47p::hTDP-43(mutant M337V)::unc-54utr transgenic. Transgenesis requires MOSSCI plasmid inserted with unc-47p::hTDP- 43::unc-54utr or unc-47p::hTDP-43(mutant M337V)::unc-54utr. Injection mix used Standard MosSCI mix. Injections were performed into mos1 ttTi5605 background strain. Extrachromosomal array lines were isolated. Crawling transgenics screened as non-red homozgotes were verified by PCR for insertion/replacement at target locus resulting verified single copy integrated strains. Transgenic <em>C. elegans</em> expressing the human TDP-43 wild-type or mutant TDP-43 M337V animal model that mimic aspects of TDP-43 specific ALS disease pathogenesis were generated. The transgenic <em>C. elegans</em> had a single copy of the human TDP-43 gene integrated into its genome. The expression is controlled by an unc-47 promoter and hence human TDP-43 protein was specifically expressed only in the <em>C. elegans</em> motor neurons. C <em>elegans </em>studies were also performed with worms transgenic for the hTDP-43 (A315T) mutation using methods described in detail elsewhere<sup>23,49</sup>.                           <strong>Age-synchronizing C. elegans                                                                                                                     </strong> Filtered deionized water is used to wash worms off of plates and into 15ml tubes which are centrifuged at 1200 rpm for 2 minutes and repeated twice. The supernatant is aspirated and 5ml of NaOH + bleach solution added. This is vortexed gently about every minute and monitored by microscope. The adults worms split open and their eggs are released. The adult worms also dissolve into the solution. Once all adult worms have dissolved, the reaction is neutralized by adding 5 ml of M9 buffer followed by three rounds of centrifugation at 2500 rpm for 2 minutes. After one wash with 10 ml of water, all but about 200- 1000ul is aspirated from the 15ml tube and the remaining pellet will be re-suspended in leftover water. This are dropped onto the plates evenly, thus ensuring that the larva that hatches have enough food while they grow over the next few days. Plates will be stored at 20°C.  <strong>Swimming-induced Paralysis (SWIP) Assay                                                                                                                          </strong> The age-synchronized worms are washed off NGM plates in S-media that contains 0.02% Triton. This allows for a more consistent number of worms while pipetting, as less worms stick to the plastic pipette tips. The volume is adjusted with S-media until there would be 60-70 worms per 20ul. Worms are scored as paralyzed if their body cannot make a bending “S” movement. Paralyzed worms can often still make small movements with their head or tail. Videos are captured using a Lumenera Infinity 3s camera fitted to a Nikon TE300 microscope at 2x magnification and recorded to ImageJ. In some experiments videos are captured using Phylumtech's Wormtracker machine. The videos will be analyzed using ImageJ <em>C. elegans</em> motility analysis software. Level of activity will be denoted based on improvement in swimming induced paralysis (SWIP) in human TDP-43 transgenic <em>C. elegans</em> disease model. The automation data measuring paralysis will measure average body bends per second of a population. Improvement in SWIP from control in the population of worms will also be observed.                                                                                                                             <strong>Drosophila Drug feeding assay</strong> Melt cornmeal-molasses-yeast fly food was mixed with certain concentrations of compound at high temperature and cooled to RT. DMSO was used as the vehicle control. Parent flies were crossed on food supplemented with drugs and the offspring were raised on the same food. Adult flies were aged on the drug-containing food for 15 days before analyzing their eye morphology. For quantification of outer eye morphological defects, ten flies were quantified. <strong>SODG93A Mouse Efficacy Study</strong> Wildtype and SODG93A mutant mice were grown for 5 weeks, then given daily IP doses with vehicle, compound T18, or compound T20 for another 5 weeks. Weight and serum pNHF were tracked during the study.
description: # Protein Assembly Modulation: A New Approach to Amyotrophic Lateral
 Sclerosis (ALS) Therapeutics
 [https://doi.org/10.5061/dryad.fqz612k0k](https://doi.org/10.5061/dryad.fqz612k0k) **Overview:** The data for this paper is available in two files: one PowerPoint and one Excel file. The PowerPoint contains scans of western blots and the excel file contains the raw data used in the paper.   **Power point file description:** Each slide in the PowerPoint contains scans of the western blots that were quantified and plotted in figure 5. Each lane of the western blot is labelled numerically at the top of the gel and a key is provided on the right side of the gel, detailing what sample is present in each lane.    **Excel file description:** Each tab in the Excel file contains the raw data corresponding to a specific figure. Tabs are labeled according to the figure number, with supplemental figures indicated by an 's' before the number. In this Excel file, cells containing "n/a" represent missing or unavailable data for that entry or indicate that the standard deviation or error could not be calculated due to the presence of only one replicate. **Excel tab descriptions:** **Figure 1** 1A- shows quantitation of stress granule reduction in PDFs following treatment with compound. In the stress granule reduction assay, PDFs were dosed with compound or vehicle for 24 hours then treated with 500uM sodium arsenite for one hour. Arsenite was then washed off and cells were fixed, permeabilized, and immunostained for TDP-43, HuR, and DAPI. Cell profiler imaging was used to calculate the number of TDP-43 positive HuR aggregates per cell under each condition and the average values and SEM are shown in the excel file. 1D- shows side-by-side comparison of values from the quantitation of the stress granule reduction assay, the nucleocytoplasmic relocalization assay, and activity against infectious HIV for chemical analogs within the THIQ series. In the excel file, the stress granule reduction and TDP-43 relocalization values are percent of control and EC50 against HIV values represent calculated EC50. **Figure 2** 2A- shows rescue of *C. elegans *transgenic for the TDP-43 A315T mutation in the SWIP assay. Worms were grown in the presence of 0.5uM THIQ compound T6, 40uM pimozide, or nothing and movement was recorded by video. The experiment was performed in 6 replicates of 25 worms each and the average number of body bends per minute and standard error is in the excel. 2B- shows rescue of *C. elegans* transgenic for the TDP-43 A315T mutation in the long term neurodegeneration assay. Worms were grown in the presence of 0.5uM THIQ compound T6, 40uM pimozide, or vehicle for 9 days and analyzed for motor neuron splits. Percentage of neurons showing splits for each replicate in each condition is in the excel. **Figure 3** 3B- T6, T11, and T12 were tested alongside vehicle in wildtype and transgenic *D. melanogaster *overexpressing C9orf72 30 G4C2. The excel shows the degeneration score observed in drosophila eyes. 3C- shows number of +(GGGGCC) and -(GGGGCC) flies in F1 generation, percent of +(GGGGCC) and -(GGGGCC) flies in F1 generation, and survival rate of C9 flies. **Figure 4** 4A- shows phosphorylated neurofilament heavy chain subunit measured in plasma of the survivors-only mice. 4B- shows mean weight change relative to baseline weight taken at week 5 over the course of the study for each mouse at each time point. **Figure 5** 5A, 5B, 5C- shows average optical density of PDI western blot and standard deviation. **Figure 6** 6- this Excel file contains a dataset of proteins identified by MSMS, organized with each row representing a protein which is labeled in column B. Columns D through G show spectral counts for these proteins for various experimental conditions. Column H indicates whether a protein is encoded by a causative gene, column I indicates whether the protein is associated with disease modifier genes, and column J lists proteins known to interact with ALS-related proteins (0 = no, 1 = yes), as per the study by Dervishi et al. (Sci. Rep. 8, 14732, 2018). Column L provides the difference in spectral counts between columns E and G. Columns M through O categorize the proteins based on the difference between WT and SOD mutation: unchanged, upregulated, or downregulated, respectively. **Supplemental figure 1** s1B- shows HIV titer (percent of control) for MT-2 cells were infected with NL4-3 Rluc HIV and treated with PAV-073 or vehicle for four days. s1C- shows observed motion of of transgenic *C. elegans* expressing the human TDP-43 A315T mutation treated with pimozide or PAV-073.