Expression constructs

The P-LoopΔ-SETX construct was generated using the Gibson Assembly Cloning Kit (NE BioLabs™) to create an eight amino acid deletion (1963–1970) of the SETX GTP/ATP binding domain (GPPGTGKS) as described recently [7]. A range of expression plasmids to study DPR toxicity were kindly provided by the laboratory of Zheng Ying, including GFP; GFP-GA30; GFP-GP30, GFP-GR30, GFP-PA30 and GFP-PR30 [45]. The GFP, GFP-GR30 and GFP-GA30 synthetic constructs were subcloned into the pSico lentiviral vector for efficient transduction studies, Sanger sequencing analysis was performed to confirm sequence integrity, and visual inspection of trial infections confirmed translation of GFP-fusion protein. Lentivirus particles were produced by triple transfecting pMD2.G, R8.74 and GFP-GR-30, GFP-GA30 or empty vector into HEK293 cells according to standard protocol. For siRNA knockdown experiments, HEK293 cells were transiently transfected for 48 h with a predesigned Silencer Select siRNA to SETX (s22951) or a Scramble control (si-CRL), using RNAiMAX™ Reagent (Invitrogen) at a final concentration of 10 nM.

Cell culture studies

Primary cortical neurons (PCN) were cultured from dissociated cortex of postnatal day-0 to day-2 (P0-P2) C57BL/6 J pups as described previously [52]. Dissociation was performed using Trypsin (T9935), Trypsin Inhibitor (T6522), and DNase I (Roche). Primary neurons were seeded onto plates coated with (0.1 mg/mL) poly-D-lysine hydrobromide (P1024) and grown in complete media (CM) consisting of Neurobasal-A medium (Thermo Fisher Scientific) supplemented with 0.5 mM L-glutamine (Thermo Fisher Scientific), 0.25% penicillin–streptomycin (Thermo Fisher Scientific), and 0.25% B-27 supplement (Thermo Fisher Scientific). PCN were maintained until treatment by removing half of the CM and replacing it with fresh CM on day 3 (where day 0 is the day of cell seeding), day 5, and every second day after day 5.

To quantify cell death, we employed a Propidium Iodide (PI) exclusion assay and a lactate dehydrogenase (LDH) cell death assay. PI is a membrane impermeable stain and therefore does not enter viable cells with intact membranes. When PI does gain access to nucleic acids and intercalates, its fluorescence increases dramatically and is therefore used to identify dead cells. To perform this assay, we removed media from each sample being careful not to dislodge adherent cells and did not rinse with PBS. We then incubated in replacement media at 37 °C containing PI (1:3000) and Hoechst dye (1:10,000) for 15 min before imaging under a Zeiss 780 LSM confocal microscope at 10X objective magnification. Briefly 3 images were taken per well and each experiment was performed in at least triplicate. The lactate dehydrogenase (LDH) assay was conducted using LDH-Cytotoxicity Assay Kit II (BioVision, K313-500-2) according to the manufacturer’s instructions. Briefly, LDH Assay Buffer and WST Substrate Mix were added to media harvested from primary neurons. Plate readings were taken at four time points: 30, 60, 90, and 120 min. The percent of LDH released was calculated as the percent cytotoxicity normalized to the amount of total protein per well (μg/μL). Amount of protein was separately determined using BCA assay using the Pierce BCA Protein Assay Kit (Cat#23225). High control cells were treated with 10 μl of the cell lysis buffer provided with the LDH assay kit and kept in the incubator for 20 min before the media was harvested for LHD assay. For Low Control the cells/neurons were treated with complete media for neuronal growth.

Western blot, immunoprecipitation and FRAP assayWestern blot analysis

Protein lysates from whole brain, spinal cord tissue or cell line extracts were prepared as previously described [43]. We loaded 30–50 µg of homogenized proteins per lane, and after running 3–8% Tris–Acetate gels (Invitrogen), samples were transferred to PVDF membranes (Millipore), which were blocked in 3% milk in PBS at RT for 1 h. Membranes were incubated with an anti-SETX Ab (A301-105A, Bethyl), anti-Flag Ab (F1804, Sigma), β-actin (ab8226, AbCam), and eGFP (A-11122, Invitrogen) in PBS-T with 3% BSA at 4 °C overnight. The primary antibody was visualized with horseradish-peroxidase conjugated anti-rabbit or anti-mouse IgG (Santa Cruz) at 1:5000 dilution and enhanced chemiluminescence (Amersham). Densitometry analysis was performed using the NIH ImageJ software application and normalized to β-actin signal intensity.

Immunoprecipitation

Cells were rinsed twice with ice-cold PBS and lysed in ice-cold lysis buffer (25 mM HEPES–KOH pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-10040 mM, one tablet of EDTA-free protease inhibitors (#11873580001 from Roche) per 10 mL of lysis buffer. The soluble fractions from cell lysates were isolated by centrifugation at 12,000 rpm for 11 min in a microfuge. Protein lysates were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific) following the manufactures protocol. For immunoprecipitations, primary antibodies were incubated with Dynabeads® (Invitrogen) overnight, and then washed with sterile PBS. Antibodies bound to Dynabeads were then incubated with lysates with rotation for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer. Immunoprecipitated proteins were denatured by the addition of 20 µl of sample buffer and heated for 10 min at 70 °C, resolved by SDS-PAGE, and analyzed via Western blot analysis.

Fluorescence recovery after photobleaching (FRAP)

HEK293 cells were transfected with three constructs (BFP or BFP-GR50, GFP or SETX-GFP, mCherry-Nucleolin), and grown for 48 h in 1% FBS in DMEM to slow cell division. Cells were imaged on a Nikon Confocal (A1 HD25/A1R HD25). Cells were chosen with clear expression of all three fluorophores, distinct nucleoli, and expression of GR50 in the nucleolus if expressed. High levels of SETX are associated with diffuse Nucleolin, likely due to S-phase arrest [7]. Thus, such cells were excluded from analysis. Nucleoli were bleached and recovery was recorded every 2 s for 90 s. 40 cells per condition were imaged, and any cells whose focus shifted during the imaging time were discarded. FRAP was calculated by correcting for background measurements, normalizing to a region of the nucleoplasm, and setting the initial bleaching reading to zero. We did not bleach the entire nucleolus as FRAP rate does not appear to be dependent on the size of the bleached region. Bleaching a larger region (an entire large nucleolus ranging from 0.5 to 3 μm in diameter) reduces the level of fluorescence intensity at the maximum recovery. Results were fit to a curve using the One Phase Association function in Graphpad Prism. Plateau values were used to determine mobile fraction, and K and half-time values used to determine dynamics. These values were analyzed by one-way ANOVA and pairwise comparisons.

Drosophila studiesUAS-Gal4 expression of wt-SETX and L389S-SETX in Drosophila

Using standard breeding strategy and maintaining flies at 25 °C, we crossed the UAS-Flag(SETX)-wt and UAS-Flag(SETX)-L389S with a range of tissue specific and developmentally staged Gal4 drivers. Lines used in this study were obtained from the Bloomington Stock Center or as described previously (Joiner, UCSD). The lines tested were as follows: vGlut-GAL4/Cyo; Cha-Gal4; D42-Gal4; tim-Gal4; Elav-Gal4; Pdf-Gal4; tsh-Gal4; 24B-Gal4; Gad-Gal4/Cyo; Ppk-Gal4; OK371-Gal4, Tdc-Gal4; Ddc-Gal4; and TH-Gal4.

Generation of SETX transgenic flies

We sought to generate SETX Tg-flies with the UAS/Gal4 based system which allows for tissue specific expression. We utilized existing SETX plasmids with N-terminal, flag-tags for expression of the wt and L389S human SETX cDNA sequences. Our Flag-wt-SETX and Flag-L389S-SETX expression constructs, described in detail previously [14], were used as the core for generating fly constructs by cloning the full length open-reading frame into the fly pUAST-attB vector (Drosophila Genomic Resource Center). Purified plasmid DNA for wt-SETX, L389S-SETX, and P-LoopΔ-SETX was submitted to the BestGene, Inc. < www.thebestgene.com > for injection for directed integration into the loci 68A4–attP2 site. The results were that n = 8 founders each for of UAS-Flag(SETX)-wt, and UAS-Flag(SETX)-L389S were generated and were subsequently outcrossed into the w1118 iso31 isogenic background for at least two generations. These were bred onto the Bristle stubble inversion balancer (Tm6b) to prevent recombination. As our analysis found that there were no detectible differences (data not shown), we now maintain just single lines of the wt-and L389S-Tg flies. The (GR)1000 lines with nSYB-GAL4 controlled pan-neuronal expression were generated as previously described [50].

Ex vivo immunohistochemistry

Drosophila salivary gland dissections were performed as described previously [50]. Larval salivary glands were dissected in PBS and fixed for 7 min in 3.7% formaldehyde in PBS. GR50 was labelled with anti-GFP (1:1000, rabbit, abcam, ab290, preabsorbed against Drosophila embryos, RRID: AB_303395). SETX-Flag was labeled with anti-Flag (mAb 1:1000, Sigma). Secondary antibodies were anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:200, RRID: AB-2338046, goat) and anti-mouse IgG (H + L) Cy3 (1:200, RRID: AB-2338685, goat). Tissues were mounted in Vectashield Hardset mounting medium (RRID: AB-2336787). Imaging was performed using a Leica DM6000 B Microscope (low magnification) or a Leica TCS SP8 Microscope (high magnification); and using a Hamamatsu ORCA-R2 C10600-10B-H camera.

NMJ analysis

Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analyzed as described previously [49, 50]. All NMJ analysis was performed double-blind. Primary antibodies used were Cy3-Conjugated anti-HRP (Goat, 1:200, Jackson ImmunoResearch Labs Cat# 123-165-021, RRID:AB_2338959) and, anti-synaptotagmin (Rabbit, 1:2000, Syt-91, RRID:AB_271399). Secondary antibodies used were anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:1000, RRID:AB_2576217, goat). NMJs were imaged for quantification using an EVOS M5000 microscope. NMJ images shown were imaged using a Leica SP5 confocal microscope. NMJ bouton number and muscle surface area was quantified manually using images in ImageJ. Bouton number and length were normalized to muscle surface area. NMJ lengths were measured from stacked NMJ images using the NeuronJ plugin for ImageJ as described previously [49, 50].

Motor climbing assay

Flies were placed individually, without anesthetization, inside glass boiling tubes mounted on a white background. After acclimatization, the flies were banged down to the bottom of the tubes to elicit the startle-induced negative geotaxis escape behavior. Videos were recorded to determine the distance in millimeters travelled in 20 s.

Studies of iPSC motor neurons

Fibroblasts were generated from dermal biopsies from ALS4 patients, upon informed consent and in compliance with IRB protocols. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 5% penicillin and streptomycin. Low passage fibroblast cultures were reprogrammed into induced pluripotent stem cells (iPSCs) and fully characterized, as done previously in our lab [47]. Isogenic SETX corrected WT and SETX KO lines were generated from an ALS4 SETX L389S line by Applied StemCell and fully characterized for clonality and chromosome integrity. Induced pluripotent stem cells (iPSCs) were maintained in mTeSR (STEMCELL Technologies, 100-0276) and were cultured at 37 °C and 5% CO2. On day 0, 1 × 106 iPSCs were seeded in NB media (DMEM/F12 with 15 mM HEPES, 200 μM Ascorbic Acid, 1X GlutaMAX, 1 N2, and 1X B27 Plus) supplemented with 1 μM Dorsomorphin, 10 μM SB431542, 3 μM CHIR99021, and 10 μM Y-27632 in one well of a 6 well plate coated with Matrigel. Media minus Y-27632 was changed daily until day 6. On day 6, cells were split 1:10 and plated in NB media supplemented with 1 μM Dorsomorphin, 10 μM SB431542, 1.5 μM Retinoic Acid, and 200 nM Smoothened Agonist, and media were changed daily until day 18, at which point cells were dissociated and re-plated on poly-orninthine/laminin coated 10 cm2 plates at 6–12 million cells/plate in NB media supplemented with 1.5 μM Retinoic Acid, 200 nM Smoothened Agonist, and 8 ng/ml each BDNF, GDNF, and CNTF. On day 24, immature MNs were re-plated for a final time in NB media supplemented with neurotrophic factors and 2 μM DAPT at a density of 250–300 × 103 cells per cm2 on poly-ornithine/laminin coated plates. On day 26, media was changed to MN maintenance media (NB with neurotrophic factors) to wash out DAPT. Half-changes of media were performed every 2 days until mature MNs were analyzed, day 31 or older.

iPSC-derived motor neurons (iPSC-MNs) were plated onto a 96 well plate with a polymer bottom black frame (Cellvis, P96-1.5P) and transduced with lentivirus encoding mCherry-NCL and GFP-GR30 for 100 h. iPSC-MNs were maintained at 37 °C with 5% CO2 during FRAP analysis. FRAP was performed using a Nikon A1R confocal microscope. FRAP conditions were empirically established such that > 85% of the mCherry-NCL signal was ablated, and neurons remained viable. After five initial scans, photobleaching was performed with the 561 nm laser at 31% power for 6 iterations. During the recovery phase, we imaged every 4 s for 100 cycles. The percentage of fluorescent recovery per minute was calculated for n ≥ 9 iPSC MNs per biological replicate (n = 3), averaged over three experiments using NIS-Elements AR analysis software.

Statistical analysis

All data were prepared for analysis with standard spread sheet software (Microsoft Excel). Statistical analysis was done using Microsoft Excel, GraphPad Prism 9.0, the VassarStats website (http://faculty.vassar.edu/lowry/VassarStats.html), or One-Way ANOVA calculator website (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). For ANOVA, if statistical significance was achieved (P < 0.05), we performed post hoc analysis to account for multiple comparisons. All t-tests were two-tailed. The level of significance (alpha) was always set at 0.05.