Mouse microglial (BV2) and motor neuron like (NSC-34) cells were stably transfected with pGL4.32[luc2P/NF-κB–RE/Hygro] plasmid DNA (Promega, Madison, WI, USA). The vector expressed 5 copies of NF-ĸB response element that drives transcription of the luciferase reporter gene luc2P. These cells were cultured in Dulbecco’s Modified Eagle Medium 16 (DMEM) supplemented with 10% fetal bovine serum. For clonal selection of the transfected plasmid these cells were cultured in 100 μg/ml hygromycin. HEK 293 cells were also used and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. The culture media for all the cells, we added 100 units/ml penicillin-streptomycin.
Evaluation of Luciferase activity assay
To measure p65 luciferase activity, BV2 or NSC-34 cells (5×104 per well) were seeded in 24-well plates. The BV2 or NSC-34 cells were stimulated with 500 ng/ml bacterial lipopolysaccharide (LPS) or 40 ng/ml of TNFα respectively. In BV2 cell experiment, after 2hrs, the media was removed and new media containing LPS with or without IMS-088 or withaferin-A was added for next 2 hrs. Similarly, for NSC-34 cells after 4 hrs of TNFα incubation, media was replaced with media containing TNFα with IMS-088 or withaferin-A for 2 hours. Post-treatment, the media was removed, and cells were gently washed with 1× PBS, and then lysed using Glo Lysis buffer (Promega, Madison). Luciferase activity was measured using the Bright-Glo Luciferase assay system (Promega,Madison), according to the manufacturer’s instructions. DMSO treated cells under similar conditions served as control. Results were expressed as mean of luciferase activity/μg of total protein from at least 3 wells in each treatment condition for BV2 cells and 4 wells of each treatment for NSC-34 cells.
Evaluation of Cell Survival
BV2 and NSC-34 cells were seeded onto 24-well plates at a density of 5X104 cells/well. The treatment paradigm was similar as explained for luciferase assay. Post incubation with the drug, cell viability was measured using MTS assay using [3-(4,5- dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium],as per the manufacturer’s instructions (Promega, Madison). The absorbance of the formazan adduct formed was determined at 490 nm using an EnSpire 2300 Multilabel reader (Perkin Elmer, Waltham, MA, USA). Values were expressed as arithmetic measurement unit.
Inhibition of Autophagosome/Lysosome pathway
Bafilomycin A1 (Sigma-Aldrich, USA), an inhibitor of Autophagosome/Lysosome pathway was used to test the effects of autophagy inhibition on IMS-088-mediated reduction of TDP-43 aggregation. HEK 293 cells were treated for 3 hrs with 50 µM Ethacrynic acid (in serum free media). Then, Bafilomycin A1 (300 nM) with or without IMS-088(1 µM) was added to media for 6hrs. After treatment, analysis of insoluble, or soluble hTDP-43 was done with 6M urea and RIPA buffers respectively followed by immunoblotting.
Sample preparation and Immunocytochemistry
HEK-293 cells were treated with 50 µM of Ethacrynic acid (Millipore sigma) overnight with or without 1 µM IMS-088.Post-treatment, cells were washed with PBS, fixed in 4% PFA for 10 minutes followed by permeabilization with 0.1% Triton X containing PBS (PBST). After permeabilization cells were washed and blocked with 10% goat serum, followed by incubation in primary antibody against hTDP-43 (1:1000) at room temperature overnight. Next morning, the cells were washed with PBST and incubated with the fluorochrome-conjugated secondary antibody (1:500) for 1h at room temperature in dark. After secondary antibody incubation, cells were washed and incubated in DAPI for 60 seconds. Following five washes cells were mounted and observed under a fluorescence microscope (Zeiss, Germany).
Mouse models and drug treatment
Transgenic mice expressing hTDP-43A315T or hTDP-43G348C were used for the experiment. Randomly selected animals were used for IMS-088 or vehicle treatment groups. IMS-088 was developed and generously provided by IMSTAR therapeutics, Canada. The average age of the mice at the start of the treatment was one year (pathological stage). 30 mg/kg of body weight IMS-088 (by gavage) was given twice a day for 8 weeks. Animals in the vehicle group received equal volumes of buffered saline for the same durations. Post-treatment, we blindly split each group into different subgroups and used all the mice for further experiments (SI Appendix; Table 1). The Animal Care Ethics Committee of Université Laval approved all in vivo experimental protocols. Experiments were carried out in accordance with the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care.
Passive avoidance and novel object recognition test
For passive avoidance test, the mice were first conditioned in a light-dark chamber with getting foot-electric shock when they enter in the dark chamber. Next day, one-trial passive avoidance was performed as described earlier (20) to check whether they avoid entering the dark chamber based on their memory function. The latency time for mice to enter the dark compartment was measured for the final test, with a cut-off time of 300 seconds.
Novel object recognition is a 3-day test, as previously described (21), in a 20 × 50 × 30 cm Plexiglas box for 5 minutes per session. Briefly, on the first day of trial, the mice were kept it in the empty box to familiarize with the environment. On the second day, two similar objects were placed in the box for mice to familiarize with the objects. On the final day of test, one of the objects was replaced with a new object and then the time spent by mice on the new object was measured for the different groups to compare the cognitive performance.
Immunohistochemistry and Image analysis
Mice were anesthetized by intraperitoneal injection using 10 μl/gm pentobarbital 12 mg/ml. The animals were transcranial perfused with ice cold phosphate buffered saline and 4% Paraformaldehyde. Post-perfusion brains and spinal cords were excised and post-fixed overnight in 4% PFA at 40C. For cryoprotection brains and spinal cords were kept in 30% sucrose solution at 40C for a day and 25µm sections were cut using a sliding VT 1200S vibratome (Leica Microsystems). Further the sections were mounted on the glass slides to procced with immunohistochemistry.
Section containing slides were washed three times with PBS (5 min) at room temperature and then antigen retrieval was performed for 20 min at 980C using 6M sodium citrate buffer. Slides were washed with 1X PBS after to room temperature. The sections were then washed in 0.25% Triton X-100 in PBS (PBST) (3 X 5 minutes) and then blocked for 1 hour in 10% goat serum in PBST in at room temperature. The sections were then incubated with primary antibodies overnight at room temperature. Next morning the sections were washed (3x10 min) with 0.25% Triton X-100 in PBS and incubated with desired secondary antibodies for 90 minutes at room temperature in a dark chamber. Prior to mounting sections were incubated with DAPI (1 min), treated with true black (1 minute) and then washed (3x10 min) with 0.25% Triton X-100 in PBS. Mounting was done onto glass slides using mounting media.
Fluorescent signal was detected using LSM 700 inverted confocal microscope (Zeiss) or Apotome (Zeiss). For each experiment, the best acquisition parameter has established considering the sections are not overexposed and the saturated signals can be avoided.
For quantifying mean fluorescence intensities of iba1 signals, at least 5-7 images per mice were captured at 20X using z-stack imaging method with Zeiss confocal microscope. We considered hippocampus and cortex separately as region of interest for analysis. Further, the quantification was performed using ImageJ software with the maximum intensity projection image. The average values were compared between saline and drug treated group. The data was represented as mean + standard error of mean (sem.)
DNA constructs, generation of transgenic mice and genotyping
The DNA construct used for the generation of the NFL-HA-mRFP1-RPL10a transgenic mice were prepared as following. First, HA-mRFP1 fragment was obtained by PCR using the following primers: 5’ primer: 5’-GGG ACG ACG AAT TCG GAG GCA GCA TGT ACC CAT ACG ATG TTC CAG ATT ACG CTG CCT CCT CCG AGG ACG T-3’ and 3’ primer: 5’-GGG ACG ACG GAT CCG GCG CCG GTG GAG TGG CGG CCC-3’.
Then, the amplified fragment was introduced into pBluescript KS+ plasmid into corresponding restriction sites. A 2.5 Kb BamHI /NotI fragment corresponding to the genomic DNA of 60s ribosomal protein L10a (RPL10a) was introduced into corresponding restriction sites of pBSKS-HA-mRFP1 recombinant vector. A 3.4 Kb XhoI/XhoI fragment corresponding to the HA-mRFP1-mRPL10a transgene was introduced into pSKhNF-L plasmid instead of the exon 1. As described in Charron G. et al, 1995 (22), this plasmid contains human NF-L gene including -292 bp of 5’ flanking sequences and intron sequences sufficient to drive NF-L expression in the nervous tissues of adult transgenic mice. To facilitate the digestion of the transgene, a KpnI restriction site was added to the pSKhNF-L plasmid at the position 6711bp.
The integrity of the final construct was verified by sequencing.
For microinjection, a KpnI-KpnI DNA fragment of 9.0 kb was isolated on agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen #28115). The transgenic mice NFL-HA-mRFP1-RPL10a named NFLrRFP were viable, did not develop overt phenotypes and were genotyped by PCR amplification. For genotyping, a 179 bp fragment from the mRFP1 gene is amplified from the NFLrRFP transgenic mice and not from the wild type mice. The PCR was performed on ear punch samples using the 5’ mRFP1-GEN primer: 5’-GACCGCCAAGCTGAAGGTGA-3’ and the 3’ mRFP1-GEN primer: 5’-CCGTCCTCGAAGTTCATCAC-3’.
The experiments presented in this paper were obtained by using the double transgeni mice named NFLrRFP;hTDP-43 A315T generated by crossing the NFLrRFP transgenic mice with our TDP43 A315T mice (supplementary figure 1B).
All experimental procedures were approved by the Laval University animal care ethics committee and are in accordance with The Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care.
Protein extraction and immunoblotting
Both tissue and cell samples were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Sigma, USA). Protein concentration was measured using Bradford reagent (Sigma, USA). 20-30 μg of protein samples were loaded and separated using SDS-PAGE followed by wet transfer on a methanol charged PVDF membrane. The membrane was blocked using 5% Bovine serum albumin for an hour at room temperature and incubated with primary antibodies overnight at 40C. The primary antibodies used at 1:1000 concentration were mouse monoclonal hTDP-43 (Abnova, Taiwan), LC3 (Novus Biologicals, USA), Beclin-1 (Novus Biologicals, USA), p62 (Millipore, USA), ATG-5 (Millipore, USA), and Actin (Millipore, USA), GFAP (Cell signalling technologies, USA), . Subsequently, the blots were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. Moreover, the blots were developed using ECL detection reagents and visualized with a StarBright Blue 520 (Bio-Rad Laboratories, USA). All band intensities were quantified using the ImageJ lab software. The membranes were incubated with suitable peroxidase conjugated secondary antibodies (Vector Laboratories, USA). Once the incubation was over, PBST wash was given to the blots. Blots were then treated with ECL reagent and developed using UNITECH imaging system (Cambridge) from Millipore, CA USA.
TRAP protocol described by Heiman and colleagues with small modifications was used for the study (23, 24). Cortex and hippocampal tissue were extracted from the brain and homogenized (10% w/v) together in tissue lysis buffer. The samples were centrifuged at 2,000 g for 10 min at 40C. Post-centrifugation, 1/9 sample volume of 10% NP-40 and 1/9 sample volume of 300 mM DHPC were added to the supernatant. Further the sample was gently mixed and then incubated for 30 min at 40C on orbital shaker. Post-incubation, centrifugation at 20,000g for 10 min was given at 40C. The supernatant was further collected and divided into two equal volume parts (one part was used for mRNA isolation and the other for peptide extraction). Each sample was added to the anti-RFP agarose affinity resin and incubated overnight at 40C on orbital shaker. Next day, the beads were isolated using centrifugation and washed 4 times using high-salt buffer (20 mM HEPES-KOH [pH 7.3], 350 mM KCl, 12 mM MgCl2, 1% NP-40, 0.5 mM DTT, and 100 mg/mL cycloheximide. The beads pellet was used either for mRNA purification or peptide purification
Purification of mRNA after TRAP Protocol
After the last washing, the beads were resuspended in 100 µL Nano prep lysis buffer with beta-mercaptoethanol for 10 min at room temperature. The RNA isolation was performed according to the kit manufacturer’s instructions (Absolutely RNA Nano prep kit). Three biological replicates were performed for each experiment (for each replicate, n = 2). Purified isolated RNA was subjected to Affymetrix mouse gene chip.
Purification of peptides after TRAP Protocol
At the end of washing, beads were resuspended in EDTA-elution buffer (10 mM HEPES-KOH [pH 7.3], 150 mM KCl, 5 mM MgCl2, 20 mM EDTA, and protease inhibitors) and incubated for 30 min at room temperature on orbital shaker. EDTA elution buffer was used to dissociate ribosomes and release nascent chain peptides. Eluate was recovered by centrifugation at 7,000 rpm for 15 min. Collected ribosome associated peptides were sequenced by mass spectrometry using Orbitrap fusion mass spectrometer. Three biological replicates were performed for this experiment (n = 6 per condition).
Prism 5.0 software (GraphPad, La Jolla, CA, USA) was used for all statistical analysis. Comparisons between 2 groups were done by unpaired two-tailed t test. Comparison between multiple groups was done by 1-way analysis of variance with Bonferroni’s or turkey post-test. A p-value up to 0.05 was considered significant.