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.

In vitro studies

Stress granule aggregation in SH-SY5Y cells overexpressing TDP-43

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.                                           

High-Content Imaging of Endogenous TDP-43 Stress Granule (SG):

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.      

Nucleocytoplasmic assay in FTD PDFs:                                                                  

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.         

Automated Image Analysis and Machine Learning Tools:                                                                                             

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.                                                                                                                                                                     

HIV infectious virus assay

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^o 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^o 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.

Drug Resin affinity chromatography

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^oC. 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^o C or 22^o 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^oC then 4^oC in 100ul column buffer containing 100uM of the cognate compound. Eluates were run on western blot or sent for mass spectrometry for analysis.

Chemical photocrosslinking

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.

Western blotting

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^oC 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.

Tandem mass spectrometry

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^o C followed by alkylation with 5o mM iodoacetamide at room temperature. Samples were then digested with trypsin (Promega) overnight (18 hours) at 37^o 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.

In vivo studies

Transgenic Human TDP-43 mutant C. elegans                                                                   

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 C. elegans 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 C. elegans 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 C. elegans motor neurons. C elegans studies were also performed with worms transgenic for the hTDP-43 (A315T) mutation using methods described in detail elsewhere^23,49.                          

Age-synchronizing C. elegans                                                                                                                    

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. 

Swimming-induced Paralysis (SWIP) Assay                                                                                                                         

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 C. elegans motility analysis software. Level of activity will be denoted based on improvement in swimming induced paralysis (SWIP) in human TDP-43 transgenic C. elegans 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.                                                                                                                            

Drosophila Drug feeding assay

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.

SODG93A Mouse Efficacy Study

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.