Research Report: Regular Manuscript Mitigation of ALS pathology by -specific inhibition of nuclear factor kappa B signaling https://doi.org/10.1523/JNEUROSCI.0536-20.2020

Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0536-20.2020 Received: 6 March 2020 Revised: 12 May 2020 Accepted: 18 May 2020

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1 Mitigation of ALS pathology by neuron-specific inhibition of nuclear factor kappa B 2 signaling 3 4 Kallol Dutta1, Sai Sampath Thammisetty1, Hejer Boutej1, Christine Bareil1 and Jean-Pierre 5 Julien1, 2. 6 7 1 CERVO Research Centre, Québec, Québec, Canada; 2 Department of Psychiatry and 8 Neuroscience, Université Laval, Québec City, Québec, Canada. 9 10 Address for correspondence: 11 Jean-Pierre Julien, CERVO Brain Research Centre; 2601, de la Canardière, Québec city, 12 (Québec), G1J2G3, CANADA. Tel: 418 663-5000 ext 4785 13 E-mail:[email protected] 14 15 Abbreviated title: Mitigation of ALS by neuronal inhibition of NF-kappaB 16 17 xNumber of pages:38 18 xNumber of figures- 11;tables- 1 19 xNumber of words for Abstract-155;Introduction- 611; Discussion-1171 20 xConflict of interest statement: The authors declare no competing financial interests. 21 xAcknowledgments: K.D. would like to thank Geneviève Soucy for her technical assistance; 22 Drs. Silvia Pozzi, Prakash K. Mishra and Karine Plourde for their critical comments and 23 discussions; animal facility technicians for care of the transgenic mice. This work was funded by 24 grants from the Canadian Institutes of Health Research, ALS Society of Canada and Brain 25 Canada. J.-P.J. holds a Canada Research Chair Tier 1 on mechanisms of neurodegeneration. 26 27 28 29 30 31

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32 Abstract 33 To investigate the role of neuronal NF-κB activity in pathogenesis of amyotrophic lateral 34 sclerosis (ALS), we generated transgenic mice with neuron-specific expression of a super- 35 repressor form of the NF-κB inhibitor (IκBα-SR) which were then crossed with mice of both 36 sexes, expressing ALS-linked gene mutants for TAR DNA-binding protein (TDP-43) and 37 superoxide dismutase 1 (SOD1). Remarkably, neuronal expression of IκBα-SRtransgene in mice 38 expressing TDP-43A315T or TDP-43G348C miceled to a decrease in cytoplasmic to nuclear ratio of 39 human TDP-43. The mitigation of TDP-43 neuropathology by IκBα-SR, which is likely due toan 40 induction of autophagy, was associated with amelioration of cognitive and motor deficits as well 41 as reduction of loss and gliosis. Neuronal suppression of NF-κB activity in 42 SOD1G93A mice also resulted in neuroprotection with reduction of misfolded SOD1 levels and 43 significant extension of lifespan. The results suggest that neuronal NF-κB signaling constitutes a 44 novel therapeutic target for ALS diseaseand related disorders with TDP-43 proteinopathy. 45 46 47 Significance statement 48 This study reports that neuron-specific expression of I-kappa B super-repressor mitigated 49 behavioral and pathological changes in transgenic mouse models of ALS expressing mutant 50 forms of either Tar DNA binding protein 43 (TDP-43) or superoxide dismutase. The results 51 suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for ALS and related 52 disorders with TDP-43 proteinopathy. 53 54 55 56 57 58 59 60 61 62

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63 Introduction 64 65 Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower 66 motor . ALS evolves with and the disease is generally fatal within 2 to 5 years 67 after the onset of symptoms. Familial ALS, accounting for around 10% of the ALS cases, is 68 caused by mutations in various genes. Expanded hexanucleotide repeats in C9orf72 account for 69 almost 40% of the familial cases whereas other mutated genes include superoxide dismutase 1 70 (SOD1), TAR DNA-binding protein (TARDBP) encoding TDP-43, fused in sarcoma (FUS) and 71 ubiquilin-2 (UBQLN2)(Picher-Martel et al., 2015). Abnormal cytoplasmic accumulations of 72 TDP-43 are a common occurrence in degenerating neurons of the central (CNS) 73 in the majority of ALS cases and of tau-negative frontotemporal dementia (FTD) as well as in 74 subsets of Alzheimer’s disease and Parkinson’s disease(Scotter et al., 2015). 75 76 Unexpectedly, TDP-43 was found to directly interact with the p65 subunit of nuclear factor 77 kappa B (NF-κB) in CNS samples from ALS individuals (Swarup et al., 2011b) and also from 78 cases with mild cognitive impairment with episodic memory deficits(Ohta et al., 2014). 79 Furthermore, we reported that TDP-43 can serve as co-activator of NF-κB in cultured 80 cells(Swarup et al., 2011b). An involvement of NF-κB in ALS is further supported by the 81 evidence that pharmacological inhibition of this pathway conferred beneficial effects in mouse 82 models of ALS including attenuation of TDP-43 pathology in motor neurons (Swarup et al., 83 2011b; Dutta et al., 2017). However, since drug inhibitors NF-κB activity can target systemically 84 different cell types it has remained unknown whether deregulated NF-κB signaling within 85 neuronal cells contributes to disease pathology and especially formation of abnormal protein 86 aggregates. 87 88 NF-κB is a ubiquitously expressed, protean eukaryotic transcription factor that is involved in a 89 multitude of physiological functions. NF-κB acts as transcription activator for several genes 90 associated with inflammation and it is considered as one of the key modulators for inflammatory 91 pathways. NF-κB remains sequestered in cellular cytosol as a complex composed of either of 92 two classes of proteins, class 1 containing of NF-κB1 (p50/p105) and NF-κB2 (p52/p100), and 93 class 2 containing RelA (p65), RelB and c-Rel. At basal state, NF-κB proteins are trapped in cell

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94 cytoplasm by association with inhibitory (I) κB proteins including IκBα, IκBβ, IκBε and Bcl-3 95 (Ghosh and Karin, 2002), their activation being dependent on a series of sequentially activated 96 kinases. When classically activated, a kinase complex (IκB-kinase or IKK complex) 97 phosphorylates IκBα on Ser32 and Ser36, leading to its ubiquitination and proteasomal 98 degradation(Viatour et al., 2005). As a result, NF-κB is released and translocates into the 99 to mediate its functions. NF-κB can undergo post-translational modifications such as 100 phosphorylation or acetylation prior to nuclear translocation. 101 102 To investigate the role of neuronal NF-κB signaling in ALS pathogenesis, we 103 generatedtransgenic mice with neuron-specific expression of a super-repressor form of the NF- 104 κB inhibitor IkappaB(IκBαS32A, S36A). The IκBα super-repressor mice were further cross-bred 105 with three different transgenic mouse models of ALS, two transgenic lines expressing human 106 TDP-43 with point mutations A315T and G348C and a mouse model expressed SOD1 with 107 G93A mutation. The hTDP-43A315T and hTDP-43G348C transgenic mice exhibit during aging 108 abnormal cytoplasmic accumulations of human TDP-43, substantial motor neuron loss as well as 109 motor and cognitive deficits (Swarup et al., 2011a). On the other hand, the well-established 110 SOD1G93Amice replicate ALS disease phenotypeswith accumulations of misfolded SOD1 111 species, severe motor neuron loss resulting in short lifespan (Dutta et al., 2018). Here, we report 112 that neuron-specific expression ofIκB-SR ameliorated behavioral and pathological phenotypes in 113 three mouse models of ALS carrying either human mutated TDP-43 or SOD1 transgenes. The 114 results suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for ALS 115 disease. 116 117 Materials and Methods 118 119 DNA constructs, generation of transgenic mice and genotyping 120 121 The DNA of H (NFH) promoter was amplified by PCR using the genomic DNA 122 purified from mouse tail. The following primers were used for the NFH amplification: 5' primer: 123 5'-GGGACGACGGTACCCTAGGACATTCTGGGCTGAGATC-3' and 3' primer: 5'- 124 GGGACGACGTCGACCAGCGGAGCGGGAGTGCGGGGCT-3'. A 2.8 Kb fragment was

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125 purified on agarose gel and subcloned into KpnI-SalI restriction sites of pBluescript KS+ 126 (pBSKS-NFH promoter). Flag-EGFP fragment was obtained by PCR using pEGFP-N3 plasmid 127 as template (CLONTECH). The following primers were used for the Flag-EGFP fragment 128 amplification: 5' primer: 5'-GGGACGACCTGCAGGAGGCAGCATGGACTA 129 CAAAGACGACGACGACAAGGTGAGCAAGGGCGAGGA-3' and 3' primer: 3'-GGGACGA 130 CGGATCCCTTGTACAGCTCGTCCATGC-3'. The obtained fragment was introduced into 131 corresponding restriction sites of pBSKS-NFH promoter plasmid. The human IκBα (SS32, 132 36AA) cDNA was amplified by PCR using the plasmid PCMV4-3HA/ IκBα (SS32, 36AA) 133 (Addgene plasmid # 24143) (Sun et al., 1996) as template. The following primers were used for 134 the PCR amplification: 135 5' primer: 5'-GGGACGACGGATCCTTCCAGGCGGCCGAGCGCCCCCAGG-3' and 136 3' primer: 5’-GGGACGACGCGGCCGCTCATAACGTCAGACGCTGGCCTCCA-3'. 137 A 953 bpBamHI /NotI fragment corresponding to the human IκBα (SS32, 36AA) cDNA was 138 introduced into pBSKS recombinant vector. The integrity of the final construct was verified by 139 sequencing. 140 For the generation of the transgenic mice, a BssHII-BssHII DNA fragment of 4.9 Kb was 141 isolated on agarose gel for microinjection. The transgenic mice were viable and did not develop 142 overt phenotypes and were genotyped by PCR amplification. For genotyping, a 329 bp fragment 143 from the EGFP gene is amplified from the NFH-IκBα (SS32, 36AA) transgenic mice (henceforth 144 referred to as IκB super repressor or IκB-SR mice) and not from the wild type (WT) mice. The 145 PCR was performed on ear punch samples using the 5' EGFP-GEN primer: 5'- 146 AGTTCATCTGCACCACCGGC-3' and the 3' EGFP-GEN primer: 5'- 147 CGGCCATGATATAGACGTTG-3'. 148 These IκB-SR mice were further cross-bred with transgenic mice bearing genomic fragment 149 expressing familial ALS-linked mutant TDP-43 (G348C and A315T) which have been 150 previously generated, characterized and reported from our laboratory (Swarup et al., 2011a). The 151 double transgenic mice, along with their littermate single transgenic and non-transgenic mice 152 have been used for this current study. The sex ratio was kept close to 1:1 for all genotype groups. 153 All experimental procedures were approved by the Laval University animal care ethics 154 committee (protocol# 2016-060-1 and 2016-060-2) and were in accordance with The Guide to 155 the Care and Use of Experimental Animals of the Canadian Council on Animal Care.

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156 Validation of IκB super repressor in vitro 157 158 To confirm the efficacy of the super repressor in inhibiting NF-κB activation, the sequence was 159 cloned into pcDNA3 vector and transfected with lipofectamine into motor neuron-like NSC-34 160 cells, seeded in 35 mm plates in duplicate (2 μg of plasmid DNA per plate). Non-transfected 161 cells from same passage served as control. At 48 h post transfection, cells were treated either 162 with recombinant mouse TNF-α (40 ng/ml) or vehicle and incubated for further 6 h. Post 163 incubations, media was discarded, the cells were harvested, lysed with RIPA buffer (10 mM 164 Tris-Cl pH 8.0; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 165 0.1% sodium dodecyl sulphate; 140 mM NaCl; protease and phosphatase inhibitors) and probed 166 for pospho-NF-κB and total NF-κB. 167 168 Motor performance test 169 170 To test motor coordination, mice were trained to run on an accelerating rotarod for one week (3 171 trials on alternate days) prior to beginning of recording. The experimental paradigm for 172 SOD1G93A, IκB-SR;SOD1G93A and their littermate controls was set at initial speed of 0-rpm with 173 acceleration of 0.1 rpm/sec whereas for the TDP-43 mutant mice, along with the double 174 transgenics with IκB-SR and their littermate controls, it was 3-rpm with acceleration of 0.2/sec. 175 The cut-off was set at 300 secs for all mice. Mice were subjected to three trials per session every 176 week and the longest latency to fall from the rotating rod was recorded. Hind limb strength of 177 SOD1G93A, IκB-SR;SOD1G93A and their littermate controls was further assessed by the grid-hang 178 test. Briefly, the mice were placed on a wire grill which was then turned over gently, allowing 179 the animal to hang from the grill. The time elapsed before the hind limbs of the mice lets go of 180 the grill was recorded with a cutoff time set at 90 seconds. 181 182 Behavioural analyses 183 184 TDP-43 pathology is also associated with fronto-temporal dementia which is replicated in our 185 transgenic animal models. To assess the effect of suppression of NF-κB activation on memory 186 functions, transgenic mice from the IκB-SR and TDP-43 mutant backgrounds (i.e. IκB-SR;TDP-

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187 43A315T, IκB-SR;TDP-43G348C, TDP-43A315T, TDP-43G348C and IκB-SR) and their non-transgenic 188 littermates were subjected to novel object recognition tests at 6 and 12 months of age. Briefly, on 189 day 1 of the test, the mice were allowed to habituate in the experimental cage for a fixed amount 190 of time (5 minutes). On day 2, two similar objects (same shape and color) were placed at two 191 pre-determined spots in the cage and the mice were allowed to explore either of them, ad libitum, 192 for the same duration of time. On day 3, one of the objects was replaced with a novel object 193 (different shape and color) and the exploratory time of the mice at both the familiar and the novel 194 object was recorded by an observer, who was blinded to the genotype of the animals. The results 195 were expressed as per cent of time spent in exploring the novel object relative to the total time 196 spent at both the objects. This test was first performed when the mice were 6 months of age and 197 repeated when they were 12 months old with entirely different set of objects. 198 199 The passive avoidance task is an aversive (emotional) conditioning paradigm whereby the innate 200 preference for the dark compartment of a test apparatus by rodents are suppressed following 201 exposure to an inescapable noxious stimuli (Ögren and Stiedl, 2010). It is a common learning 202 and memory function test that is commonly used to test for cognitive decline in models of 203 neurodegeneration. On day 1 of the test, the mice were allowed to familiarize with the 204 surroundings. Initially, the mice were placed in a lighted chamber that was separated from a dark 205 chamber by a time controlled door. After a specific time (30s) the door was programmed to open 206 and based on the natural light avoidance tendency of the mice, the time taken to enter the dark 207 chamber was noted. On day 2, the mice were allowed to remain in the lighted chamber for the 208 same duration of time but the dark chamber was rigged to deliver an electric shock (0.5 mA for 209 5s) via the footpad. Once the mice entered the dark chamber and received the footpad shock, 210 they were confined in there for a further 60s so as to reinforce the memory of the noxious 211 stimuli. On day 3 the mice were once again placed in the lighted chamber but the door 212 connecting it with the dark chamber was opened after 5s. The time taken by the mice to re-enter 213 the dark chamber was noted, again by an observer blinded to the genotypes. A significant delay 214 or refusal to re-enter the dark chamber was considered to be indicative of learned response 215 against the noxious stimuli. Due to the stressful nature of the passive avoidance task, the mice 216 were subjected to only one trial at 12 months of age. 217

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218 Analysis of clinical symptoms of SOD1G93A mice 219 220 The SOD1G93A model is an acute model of familial ALS. The onset of disease was estimated to 221 correspond with the time when mice started to exhibit a decline of body weight after reaching a 222 peak. End stage was defined as the loss of righting (the age when the animal could not 223 right itself within 30s when placed on its side or when a loss of greater than 30% peak body 224 weight was recorded). A 3-point scoring system based on hind-limb , where non- 225 symptomatic animals (hind limbs extended forming an angle of 120 degrees) was assigned a 226 score of 3 whereas end stage animals (showing loss of reflex with hind limbs paws held close to 227 the body and unable to walk) being assigned a score of 0, was used to monitor the progression of 228 disease in SOD1G93A mice (Urushitani et al., 2006). The same approach was also adopted for the 229 IκB-SR;SOD1G93A and littermate controls. This reflex scoring was done by animal facility 230 technicians who were blinded for genotypes but had experience in grading paralysis in mice. The 231 body weights of these animals were also recorded, weekly prior to advent of symptoms and daily 232 post-symptom. The decline in body weight also serves as a clinical indicator for rate of disease 233 progression in this model. 234 235 and immunofluorescent staining of tissue sections 236 237 Animals were anesthetized by intraperitoneal administration of sodium pentobarbital (10 mg/Kg 238 body weight) and transcardially perfused with ice-cold saline followed by fixation with cold 239 solution of 4% paraformaldehyde. Spinal cords were excised carefully and stored overnight in 240 4% paraformaldehyde solution. On the following day the tissues were transferred to a 30% 241 sucrose solution and stored for 24-48 hours prior to sectioning with a microtome. 242 sections (20 μm thickness) were mounted on glass slides and allowed to adhere in a vacuum 243 chamber. The slides were washed with 1× TBS (50 mM Tris-Cl, pH 7.5; 150 mM NaCl) and 244 permeablized with 1× TBS-0.1% Triton-X 100. The slides were then blocked for 2h at room 245 temperature with 10% solution of normal goat serum (Life Technologies, CA, USA) in 1× TBS- 246 0.1% Triton-X 100. While staining for FLAG using anti-FLAG M2 antibody, blocking was done 247 with a 5% solution of non-fat milk in 1× TBS-0.1% Triton-X 100, as other blocking agents (goat 248 serum or 10% albumin) failed to reduce non-specific antibody binding. Post blocking, sectioned

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249 were incubated overnight at 4℃ with appropriate primary antibodies (see Table 1 for details). 250 On the next day, the slides were washed extensively with 1× TBS followed by incubation 251 withappropriate fluorochrome-conjugated secondary antibodies (Alexa Fluor; Thermo Fischer 252 Scientific, IL, USA) at room temperature in a dark chamber, for 2h. After washes, slides were 253 briefly incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fischer 254 Scientific, IL, USA) followed by another incubation (10 minute) with 0.05% Sudan Black B 255 solution in 70% ethanol to quench autofluorescence. After repeated washes, slides were 256 mounted with Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA, USA) and 257 visualized under a Zeiss Apotome or a Leica DM5000B microscope. 258 259 Image analysis 260 261 Image analysis was performed using the FIJI software (freely downloadable from 262 https://imagej.net/Fiji). For measuring cellular fluorescence signal intensity methodology 263 described by Arqués et al was used, with minor modifications (Arqués et al., 2012). Briefly, the 264 regions of interest (ROI; cell boundary or nucleus) were marked using the freehand selection 265 tool. The area of the ROI and integrated density was measured along with the mean fluorescence 266 of background. The corrected total fluorescence (CTF) was calculated using the following 267 formula: CTF = Integrated Density – (Area of ROI × Mean fluorescence of background 268 readings). GFAP and Iba-1 signal intensities from ventral horns of spinal cord was measured by 269 method previously described (Dutta et al., 2017). Microglial branching pattern analysis were 270 performed by methods described by Young and Morrison, with minor modifications. Briefly, z- 271 stack images of spinal cord sections at 20x magnification were captured and max intensity 272 projection images were generated. The images were then adjusted for optimal brightness and 273 contrast followed by application of an ‘Unsharp mask’ (pixel radius 3; mask weight 0.6). The 274 images were then despeckled to remove ‘salt-and-pepper’ noise followed by their conversion to 275 binary images by threshold adjustment. The binary images were subjected to another round of 276 despeckling followed by outlier removal (pixel radius 2; threshold 50). Finally the binary images 277 were skeletonized and saved as separate files. At random, skeleton images of cells (at least 24 278 from 3 animals) were reopened with FIJI and run through the ‘AnalyzeSkeleton(2D/3D)’ plugin 279 and checking the ‘Branch Information’ box. The generated data were saved in an Excel

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280 spreadsheet. The data were sorted by ‘endpoint voxels’ from largest to smallest and the cut-off 281 length for undesired fragments was set at 0.5 μm. The number of branching per cell and total 282 length of branches per cell were tabulated in a separate file which were used for phenotypic 283 analysis (Young and Morrison, 2018). 284 285 Immunoblot analysis 286 287 Animals were anesthetized by method described above. Tissues (brain, spinal cord, kidney, liver, 288 spleen) were excised quickly and snap frozen in liquid nitrogen followed by storage in -80℃ 289 freezer until further use. Hippocampus from mice brain were dissected following protocol 290 demonstrated in European Journal of Neuroscience’s protocol video (retrieved from 291 EJNeuroscience’s YouTube channel https://www.youtube.com/watch?v=Upf15CB29V4). In 292 general, tissues were homogenized using chilled RIPA buffer, sonicated and centrifuged for 293 20 min at 12000 g at 4°C. The removing the supernatant (soluble fraction), the pellets were 294 washed thrice with RIPA buffer followed by resuspension in Urea (6M) buffer which served as 295 insoluble fraction. Spinal cords from SOD1mice carrying the SOD1 mutation were homogenized 296 in a lysis buffer (TGNT buffer) containing 50 mM Tris–HCl (pH: 7.4), 100 mM NaCl, 10 % 297 glycerol and 1 % Triton X, sonicated followed by centrifugation for 20 min at 9000g at 4°C. 298 299 Proteins from tissue lysates were electrophoretically separated on SDS-polyacrylamide gel and 300 transferred onto PVDF membranes. The membranes were then blocked with 7% non-fat milk in 301 1× PBS containing 0.1% Tween-20 followed by incubation with primary antibodies (see Table 1 302 for details) diluted in 1% bovine serum albumin solution. Post incubation, blots were washed and 303 re-incubated with appropriate secondary antibodies conjugated with horse-radish peroxidase. 304 Signal was acquired by exposing chemiluminescent reagent treated membranes to X-ray films 305 (Biomax MR, Kodak) or in a ChemiDoc MP Imaging System (BioRad). Band intensities were 306 analyzed using FIJI and normalized to housekeeping proteins Actin or GAPDH. To visualize 307 total proteins on gels for normalizing insoluble proteins, Coomassie staining protocol was used. 308 Briefly, after electrophoresis, gels were incubated in Coomassie stain (0.1% Coomassie R-250 in

309 50% methanol, 10% acetic acid, 40% H2O) and incubated at room temperature for 1h. The gel

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310 was then incubated in destainer (5% methanol, 7.5% acetic acid, 87.5% H2O) with changes until 311 a clear background was achieved. 312 313 Immunoprecipitation of FLAG fusion proteins 314 315 For proteomic confirmation of IκB-SR transgene expression, spinal cords from IκB-SR 316 transgenic mice were lysed in RIPA buffer supplemented with protease inhibitor cocktail. The 317 homogenates were centrifuged at 20000×g for 10 min at 4℃ and the supernatants were added 318 onto paramagnetic Protein A/G beads (Dynabeads; Invitrogen, Carlsbad, CA, USA) pre-coated 319 with anti-FLAG M2 antibody (Table 1). The sample-bead mixtures were incubated overnight at 320 4℃ on an orbital shaker. The following day, the beads were washed and samples were eluted 321 with Laemmli sample buffer and subjected to SDS-PAGE and immunoblotting using anti-IκBα 322 antibody. 323 324 Co-Immunoprecipitation for P65 NF-κB and hTDP-43 325 326 Protein A/G coated paramagnetic beads (Dynabeads; Invitrogen, Carlsbad, CA, USA) were 327 coated with anti-P65 NF-κB antibody (Table 1) at the recommended concentration (1.5 μg) for 2 328 h at room temperature. Post washings to remove unbound IgG, the beads were incubated with 329 spinal cord lysates in RIPA buffer containing 300 μg proteins, at 4°C, overnight. On the 330 following day the beads were washed, proteins eluted and separated on a 10% SDS- 331 polyacrylamide gel followed by electrophoretic transfer onto a PVDF membrane. The 332 membranes were subsequently blocked with 5% BSA and incubated overnight with anti-human 333 TDP-43 antibody. Immunodetection was done using goat anti-rabbit-horseradish peroxidase 334 labeled secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, 335 USA) and visualized on X-ray film (Biomax MR1; Kodak, Rochester, NY, USA) following 336 exposure to enzyme chemiluminecent reagent (Thermo Fischer, IL, USA). 337 338 Immunoprecipitation for misfolded SOD1 339

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340 Antibody against mis-folded SOD1 (clone B8H10) was purified from serum-free culture media 341 of hybridomas. The media was treated with a saturated solution of ammonium sulphate and the 342 salted-out proteins were subsequently dialyzed to obtain the antibody. Immunoprecipitation was 343 performed using protein A/G-coated paramagnetic beads (Dynabeads; Invitrogen, Carlsbad, CA, 344 USA) according to previously published protocol with minor modifications (Gros-Louis et al., 345 2010). Briefly, the beads were first coated with the monoclonal anti-misfolded SOD1 antibody 346 (clone B8H10; 1.0 μg of antibody per 40 μL of beads) for 2 h at room temperature. Following 347 washes to remove unbound antibodies, the beads were incubated with volumes of spinal cord 348 lysates corresponding to 100 μg of total protein, at 4°C, overnight. On the following day, the 349 beads washed, proteins were eluted and separated on a 14% SDS-polyacrylamide gel and then 350 transferred electrophoretically to PVDF membrane. After blocking with 5% non-fat milk, 351 membranes were incubated with polyclonal primary antibody against SOD1 (see Table 1). 352 Immunodetection was performed with a goat anti-rabbit-horseradish peroxidase labeled 353 secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and 354 visualized on X-ray film (Biomax MR1; Kodak, Rochester, NY, USA) following exposure to 355 enzyme chemiluminescent reagent (Thermo Fischer, IL, USA). 356 357 Statistical Analysis 358 359 All statistical analysis was done using GraphPad Prizm (version 7.01). Prior to analyzing data 360 from multiple groups, the normal distribution of the data was checked using the D'Agostino & 361 Pearson omnibus normality test. Comparisons between multiple groups with normally distributed 362 data were done using one-way ANOVA with Tukey's posttest. In cases of non-normal 363 distribution, data were analyzed using Kruskall-Wallis test along with Dunn's posttest. 364 Comparisons between 2 groups were done by Student's T test with Welch’s correction. 365 366 Results 367 368 Neuron-specific expression of IkB-super repressor 369

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370 Using genetic engineering, we generated a cDNA construct encoding in frame FLAG, EGFP and 371 human mutant IκBαS32A, S36Aunder the control of neurofilament Nefh gene promoter. The Nefh- 372 FLAG/EGFP/IκBαS32A, S36A (Nefh-IκB-SR) DNA construct was used to generate transgenic mice 373 by the microinjection of DNA into fertilized eggs followed by implantation in pseudo-pregnant 374 female mice (Fig. 1A). Four transgenic founders were initially generated (#1397, #1398, #1399, 375 #2373), which were then assessed for levels of transgene expression based on FLAG and GFP 376 detection from spinal cord and brain samples. Microscopy of immunostainedspinal cord sections 377 revealed the highest neuronal expression levels of FLAG and GFP in founder number 1397 (Fig. 378 S1A). Immunoblots of spinal cord lysates confirmed prominent detection of the IκB-SR protein 379 of about 63 kDa (IκBα + FLAG + GFP) in spinal and brain samples from #1397 380 transgenicmouse (Fig. S1B, C).As the transgene was under the Nefh gene promoter, its 381 expression was limited to the nervous tissues. To confirm this, non-neuronal tissues (kidney, 382 liver and spleen) were excised from IκB-SR transgenic mice and their lysates were subjected to 383 SDS-PAGE followed by immunodetection with anti-FLAG or anti-GFP antibodies. Immunoblots 384 revealed no signal in non-neuronal tissues for IκB-SR protein at expected 63 kDa (Fig. S1D). 385 Based on these results, a mouse colony from the founder #1397 was generated by backcrossing 386 transgenic mice in the C57BL6 background. Mice from the IκB-SRline (#1397) were then bred 387 with transgenic models of ALS expressing either mutant TDP-43 species or mutant SOD1G93A 388 (Fig. 1B). 389 390 The presence of IκB-SR transgene was confirmed at every step by genotyping (Fig. 1C). Spinal 391 cord lysates from IκB-SR and littermate WT mice were immunoprecipitated with anti-FLAG M2 392 antibody and subsequently fractionated by SDS-PAGE with ensuing immunoblotting with anti- 393 IκBα antibody. A parallel experiment using mouse IgG served as control. Results further 394 confirmed the immunodetection of IκB-SR protein by the anti-IκBα antibody in spinal cord 395 lysates from transgenics but not from WT mice (Fig. 1D). To confirm the cell specificity of the 396 transgene expression, spinal cord sections from IκB-SR mice were immunostained for FLAG 397 and GFAP ( marker) or FLAG and Iba-1 ( marker). Results clearly show that 398 none of the GFAP or Iba-1 positive cells were positive for FLAG, thereby confirming neuron- 399 specific expression of IκB-SR (Fig. 1E). 400

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401 To validate the efficacy of IκB-SR transgene expression to inhibit IκBα activation in vivoin the 402 CNS, mice were injected with LPS from E. coli intraperitoneally and after 12 h the mice were 403 sacrificed for immunodetection of phosphorylated IκBαspecies in their spinal cord and spleen. 404 In normal WT mice, LPS treatment led via activation of TLR4 pathway to substantial induction 405 of IκBα phosphorylation in both the spleen and spinal cord (Fig. S1E). In contrast, LPS 406 treatment of IκB-SR transgenic mice caused induction of IκBα phosphorylation in the spleen but 407 not significantly in the spinal cord due to expression of the NF-κB inhibitor construct IκB- 408 SR(Fig. S1E). Moreover, in vitrotransfection studies with NSC-34 (motor neuron-like) cells 409 confirmed that expression of IκB-SR attenuated levels of phosphorylated IκBα species before or 410 after stimulation by TFN-α (Fig. S2A). 411 412 Reduced nuclear translocation of p65NF-κB in spinal neurons of IκB-SR;TDP-43A315T mice 413 414 To assess the suppression of NF-κB activation in spinal neurons of IκB-SR;TDP-43A315T mice, 415 spinal cord sections were immunostained with anti-phospho p65 NF-κB antibody and 416 counterstained with anti-neuron specific antibody (NeuN) and DAPI to mark the nucleus (Fig. 417 2A-F). Using ImageJ software, neuronal nuclear area was outlined at first, followed by 418 estimation of phospho-NF-κB signal intensity within that specified region. It is noteworthy that 419 the nuclear signal intensity for phospho-NF-κB was increased in TDP-43A315T mice and TDP- 420 43G348C mice by ⁓90% and ⁓200%, respectively, as compared to WT or IκB-SR mice (Fig. 2G). 421 A significant reduction of ⁓50% in the nuclear signal of phospho-NF-κB in the neuronal nuclei 422 of IκB-SR;TDP-43A315T mice when compared to single transgenic TDP-43A315Tmice. An almost 423 equivalent reduction (⁓53%) was also measured in IκB-SR;TDP-43G348C mice as compared to 424 single transgenic TDP-43G348C. Co-immunoprecipitation studies were performed to evaluate the 425 interaction between NF-κB and TDP-43. Spinal cord lysates from TDP-43 mutant mice or from 426 the respective double transgenics with IκB-SR were subjected to immunoprecipitation with anti- 427 NF-κB antibody, proteins fractionated by SDS-PAGE with ensuing immunoblotting with the use 428 of anti-human TDP-43 antibody. Results revealed a co-immunoprecipitation of TDP-43 with 429 anti-NF-κB antibody using spinal cord lysate from in TDP-43A315T mice. In contrast, inhibition 430 of NF-κB signaling by IκB-SR prevented interaction of TDP-43 with NF-κB. Thus, no TDP-43 431 signal was detected in immunoprecipitates from IκB-SR;TDP-43 mice (Fig. 2H).

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432 Suppression of NF-κB signaling alleviates cytoplasmic accumulations of TDP-43 in spinal motor 433 neurons 434 435 Transgenic mice expressing human TDP-43 mutants exhibit age-dependent accumulations of 436 human TP-43 in the cytoplasm of spinal motor neurons. Thus, in 1 year old single transgenic 437 mice expressing either TDP-43A315T or TDP-43G348C, the anti-human TDP-43 immunostaining 438 was mainly detected in the cytoplasm of neurons in the ventral region of the spinal cord (Fig. 3B 439 and D). In contrast, human TDP-43 was predominantly localized in the nucleus of spinal neurons 440 in sections from doubly transgenic mice, either the IκB-SR;TDP-43A315T or IκB-SR;TDP-43G348C 441 mice (Fig. 3A and C). The TDP-43 signal intensity in cytoplasm and nucleus was estimated by 442 marking the cell and nuclear boundaries (cytoplasm = whole cell – nucleus) using multiple spinal 443 cord sections pooled from 3 mice per group (Fig. S2B). Results revealed a2-fold decrease in 444 cytoplasmic to nuclear TDP-43 signal ratio in IκB-SR;TDP-43A315T mice when compared to 445 single transgenic TDP-43A315Tmice (Fig. 3E). There was also a1.4-fold decrease in cytoplasmic 446 to nuclear signal ratio in IκB-SR;TDP-43G348C when compared to TDP-43G348Cmice. To assess if 447 the cytoplasmic accumulation of TDP-43 is affected by the size of the neuron, we tabulated the 448 cytoplasmic to nuclear ratio with corresponding area of neurons (with cutoff area >250 μm2). 449 The ratios in both IκB-SR;TDP-43A315T and TDP-43A315T groups correlated with increasing 450 neuronal size as calculated by Spearman’s rank correlation test (95% C.I.; ρ for Area v/s 451 C:N of IκB-SR;TDP-43A315T = 0.4520; p<0.0001; ρ for Area v/s C:N of TDP-43A315T = 0.2697; 452 p=0.0033) (Fig. 3F). However, the cytoplasmic to nuclear ratio of TDP-43 signal in IκB- 453 SR;TDP-43G348C did not significantly correlate with the neuronal soma area even though that in 454 TDP-43G348C did correlate with area (ρ for Area v/s C:N of IκB-SR;TDP-43G348C = 0.1677; 455 p=0.0575; ρ for Area v/s C:N of TDP-43G348C = 0.2519; p=0.0079) (Fig. 3G). RIPA soluble and 456 insoluble fractions of spinal cords of mice from various groups were subjected to 457 immunoblotting. Results showed that in RIPA soluble fractions of both IκB-SR;TDP-43A315T and 458 IκB-SR;TDP-43G348C mice spinal cords, human TDP-43 was present at a significantly higher 459 level as compared to respective TDP-43 mutant mice tissues (Fig. 3H&J). Contrarily, in the 460 RIPA insoluble fractions, more human TDP-43 was detected from the TDP-43 mutant mice 461 tissues (Fig. 3I&K). 462

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463 Neuronal IκB-SR expression conferred protection of motor neurons with ensuing improvement of 464 motor performance 465 466 Studies have shown that alpha motor neurons are vulnerable in ALS whereas gamma motor 467 neurons remainmostly unaffected (Conradi and Ronnevi, 1993; Lalancette-Hebert et al., 2016). 468 For estimation of alpha motor neuron numbers in spinal ventral horns, a size-based approach was 469 adopted in the current study, rather than detection with the classical marker choline acetyl 470 transferase (ChAT). ChAT is expressed in both alpha and gamma motor neurons whereas the 471 neuronal DNA binding protein NeuNis expressed in alpha but not (or poorly) in gamma motor 472 neurons (Friese et al., 2009; Shneider et al., 2009; Gusel'nikova and Korzhevskiy, 2015). Gamma 473 motor neuron cell body area ranges from 100 to 250μm2, whereas the larger alpha motor neurons 474 range from 250 to 1200μm2 (Drachman and Rothstein, 2000; Pasetto et al., 2017). In our study, 475 we have considered NeuN-positive cells in spinal ventral horn with area more than 250 μm2 as 476 alpha motor neurons. The cells were from multiple spinal cord sections chosen at random from at 477 least 3 mice per group (Fig. 4A). Results showed that there were significantly more alpha motor 478 neurons in spinal cord of IκB-SR;TDP-43A315T mice in comparison to TDP-43A315T mice (17.24 ± 479 1.573 v/s 11.47 ± 0.875). Similar difference was also observed between IκB-SR;TDP-43G348C 480 and TDP-43G348C (14.29 ± 1.007 v/s 9.783 ± 0.7724). Nonetheless, these values were still 481 significantly lower than that recorded either from IκB-SR (21.82 ± 0.613) or WT (23.52 ± 1.228) 482 mice (Fig. 4b), suggesting IκB-SR transgene expression did not completely rescue the loss of 483 alpha motor neurons in the TDP-43A315T and TDP-43G348C mice. 484 485 The motor phenotypes in these TDP-43 ALS mouse models are characterized by the progressive 486 loss neuromuscular junctions and decrease of motor performance (Swarup et al., 2011a). To 487 assess the effect of chronic inhibition of neuronal NF-κB signaling on motor performance, the 488 mice from all six genotypes were trained to run on an accelerating rotarod from the age of 120 489 days (4 months) and monitored weekly until 12 months of age. The maximum latency to fall 490 from the rotarod out of three trials per mouse per week was recorded and tabulated together (Fig. 491 4C). To elucidate the comparisons and ease of analysis, the data were split to show IκB-SR vs 492 WT, IκB-SR;TDP-43A315T vs TDP-43A315T and IκB-SR;TDP-43G348C vs TDP-43G348C groups. 493 There was no significant difference in motor performance of IκB-SR transgenic mice when

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494 compared to their non-transgenic littermates, with latency to fall of ~80 sec (Fig. 4D). In 495 contrast, during aging (starting at ~300 days of age) the TDP-43A315T mice and TDP-43G348C 496 mice exhibited decline of motor performance in the rotarod test with latency to fall within ~60 497 sec (Fig. 4 C, E, F). Remarkably, neuronal expression IκB-SR in doubly transgenic mice 498 rescued the age-dependent deficits of motor performance due to expression of TDP-43 499 mutations. Hence, the doubly transgenic IκB-SR;TDP-43A315T and IκB-SR;TDP-43G348C mice 500 exhibited latency to fall of ~80 sec in the rotarod test. There were no significant differences in 501 the body weights of mice from all six groups which could affect fluctuations in the rotarod 502 performance (Fig. S2C). 503 504 Neuronal inhibition of NF-κB signaling mitigated cognitive deficits in transgenic mice 505 expressing ALS-linked TDP-43 mutations 506 507 In a previous study we reported age-dependent cognitive declines in transgenic mice expressing 508 ALS-linked TDP-43 mutations (Swarup et al., 2011a). Here, we tested the effects of suppressing 509 neuronal NF-κB signaling in mice and subjected them to the object recognition test at 6 and 12 510 months of age. The single transgenics TDP-43A315Tand TDP-43G348C exhibited impaired 511 performance at novel object recognition with significant reduction in exploration time at novel 512 object as compared to their WT or IκB-SR littermates (Fig. 5A, B). Remarkably, neuronal 513 expression of IκB-SR transgene in mice expressing TDP-43 mutations restored their preference 514 for the novel object at a degree comparable to WT or IκB-SR mouse littermates (Fig. 5A, B). 515 At 12 months of age, passive avoidance test was also performed to assess the fear-induced 516 learning and memory capacities of the animals belonging to each mouse genotype. Results 517 showed that TDP-43G348C mice did not recollect the adverse electric shock experience and they 518 entered the dark chamber within few secs. In contrast, mouse littermates with the IκB-SR;TDP- 519 43G348C genotype performed very well with an average latency of ~200 sec, which is merely 520 below the latency mice belonging to the IκB-SR and WT groups. Somehow, no significant 521 difference in the passive avoidance test was observed between the IκB-SR;TDP-43A315Tmice and 522 TDP-43A315Tmice (Fig. 5C). The performance of these TDP-43A315Tgroups significantly differed 523 from those of TDP-43G348C, IκB-SR and WT mice.Hippocampal extracts from non-littermate 12 524 month old mice were subjected to immunoblotting to check TDP-43 levels. Results revealed

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525 higher levels of TDP-43 in hippocampal extracts from the TDP-43A315Tmice than from TDP- 526 43G348C mice. However, the proportion of detergent-insoluble TDP-43 to soluble TDP-43 was 527 more elevated in extracts from TDP-43G348Cthan in extracts from TDP-43A315T mice, which could 528 explain the discrepancy in the performance at passive avoidance test (Fig. 5D).] 529 530 Neuronal IκB-SR expression attenuated gliosis 531 532 To investigate the effects of IκB-SR on neuroinflammation, spinal cord sections from the six 533 groups of mice were immunostained with antibody against glial fibrillary acidic protein (GFAP), 534 a marker of , and with antibody against Iba1 a marker of microglia (Fig. 6A, C). 535 Immunofluorescence microscopy revealed a marked up-regulation of GFAP signals reflecting 536 astrocytosis in single transgenic mice expressing TDP-43 mutations in comparison to those from 537 doubly transgenic IκB-SR;TDP-43A315T and IκB-SR;TDP-43G348Cmice. The GFAP signal 538 intensities in IκB-SR;TDP-43A315T and IκB-SR;TDP-43G348C mice were similar to the GFAP 539 immunostaining in single IκB-SR transgenic mice (Fig. 6B). 540 541 The morphology of activated microglia is known to be extremely plastic. Between their resting 542 filamentous or hyper-ramified morphology and phagocytic amoeboid shape, microglia undergoes 543 multiple phenotypic alterations as it responds to surrounding environment (Kreutzberg, 1996). 544 Since they are finely tuned to neuronal and glial function through continuous inter-cellular cross- 545 talk and in vivo motility, quantitative analysis of microglia morphologies can serve as indicators 546 of neuropathology in CNS (Young and Morrison, 2018). Iba-1 staining was performed on spinal 547 cord sections from the six groups to visualize microglial morphology (Fig. 6C). The overall Iba- 548 1 signal intensity was significantly higher in the single transgenic mice expressing TDP-43 549 mutations when compared to the double transgenicsIκB-SR;TDP-43A315T and IκB-SR;TDP- 550 43G348C. 551 552 Surprisingly, the Iba-1 signal intensity in spinal cords of IκB-SR mice was also abnormally 553 elevated (Fig. 6D). As the microglial morphology distinctly differed in each of the 6 groups, a 554 spatiotemporal analysis was required to better understand their reactive states. The cell images 555 were converted to their binary skeleton form (Fig. 6E) which was then analyzed for branching

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556 patterns by processes described in the Methods and Materials section. The results of this analysis 557 showed that in both TDP-43A315T and TDP-43G348C mice spinal cords, there was significantly less 558 branching points and decreased branch lengths per microglial cell as compared to those observed 559 in either IκB-SR;TDP-43A315T or IκB-SR;TDP-43G348C mice. Thus, the number of branching 560 points and total branch lengths in these double transgenic mice were similar to that observed in 561 the single IκB-SR mice, indicating that the spinal cord microglia in these groups were more 562 ramified and in a relatively less reactive state (Fig. 6F, G). 563 564 Inhibition of NF-κB signaling by IκB-SR induced autophagy 565 566 Immunofluorescent staining at microscopy for LC3B, P62 and ATG5, markers of autophagy, 567 revealed higher signals in neuronal cytoplasm of spinal cord sections from doubly transgenic 568 mice co-expressing IκB-SR with TDP-43 mutations than in sections from single transgenic TDP- 569 43A315T and TDP-43G348C mice (Fig. 7 A-E). Moreover, western blot analysis of spinal cord 570 lysates revealed increased levels of beclin1, another autophagy marker, in transgenic mice 571 expressing the IκB-SR transgene (Fig 7F & G). 572 573 Neuronal expression of IκB-SR delayed disease progression and mortality in SOD1G93A mice 574 575 We have further examined the effects of neuronal suppression of NF-κB in SOD1G93A mice, 576 another model of ALS. As shown in Fig. 8A, neuronal expression of IκB-SR transgene in 577 SOD1G93A mice resulted in an increase of median survival by 15 days (171 days for IκB- 578 SR;SOD1G93A v/s 156 days for SOD1G93A). The survival was extended by more than 20 days in 579 some litters (data not shown). 580 The locomotor performance of the mice was compared on an accelerating rotarod. The IκB- 581 SR;SOD1G93A and SOD1G93A performed similarly between 63 days to 126 days of age. Then 582 there was a sharp decline in latency to fall for the SOD1G93Amice until 146 days of age. The 583 decline in performance was significantly less pronounced with IκB-SR;SOD1G93A between 126 584 and 146 days of age (Fig. 8B). The grid hang test was performed to assess hind limb strength but 585 there was no significant difference between the two mouse groups throughout their life span 586 (Fig. 8C). On the other hand, the reflex scores of doubly transgenic mice were superior to those

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587 of single SOD1G93A mice at end stage of disease starting at 146 days of age (Fig. 8 D). Loss of 588 body weight was also monitored. We found no significant difference between the actual body 589 weights of the mice belonging to either groups (Fig. 8E). However, when the data was plotted as 590 percent of maximum, the body weight of mice belonging to the SOD1G93A group peaked at 119 591 days of age whereas for IκB-SR;SOD1G93A it peaked at 126 days. Between 126 days and 147 592 days of age, there was a significant difference between the 2 groups, indicating a moderate 593 slower rate of disease progression in the IκB-SR × SOD1G93A mice (Fig. 8F). 594 595 Reduced levels of misfolded SOD1 in IκB-SR;SOD1G93A mice at different stages of disease 596 597 Accumulation of misfolded SOD1 in spinal motor neurons is a pathological hallmark of mutant 598 SOD1-mediated disease that can be detected in SOD1G93A mouse as early as 30 days of age 599 (Gros-Louis et al. 2010; Maier et al., 2018). Using an antibody (B8H10) specific against 600 misfolded SOD1, we examined the levels of mis-folded SOD1 in spinal cord extracts by 601 immunoprecipitation with B8H10 antibody followed by PAGE and immunoblotting with anti- 602 pan SOD1 antibody. In all three time points (pre-symptomatic 55 days, early symptomatic 125 603 days and end stage), the levels of misfolded SOD1 were lower (upto ⁓40% at 55 days, ⁓30% at 604 125 days and ⁓10% at end stage) in spinal cord samples of IκB-SR;SOD1G93A mice than in 605 samples from age-matched littermate SOD1G93A (Fig. 9A). However, taken together, the 606 differences between IκB-SR;SOD1G93A and SOD1G93A were not statistically significant (Fig. 607 9B). The enzyme ChAT is necessary for maintaining motor neuron functions and in ALS cases 608 these cells loose ChAT immune-reactivity prior to degeneration (Oda et al., 1995). At early 609 symptomatic stage (125 days), spinal cord neurons in IκB-SR;SOD1G93A mice retained ChAT 610 immunoreactivity unlike SOD1G93A mice (Fig. 9C). This result is consistent with the higher 611 number of motor neurons (area > 250 μm2) NeuN+ detected in the ventral horn spinal cord of 612 IκB-SR;SOD1G93A (14.81 ± 0.7173) as compared to SOD1G93A (6.485 ± 0.7883) (Fig 9D & E). 613 614 Discussion 615 Abnormal cytoplasmic aggregates of TDP-43 in degenerating neurons are being recognized as a 616 hallmark of ALS, frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related 617 TDP-43 encephalopathy (Neumann et al., 2006; Nelson et al., 2019).There are many lines of

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618 evidence suggesting that cytoplasmic aggregation of TDP-43 may play a key role in 619 neurodegeneration (Hergesheimer et al., 2019). Here we report for the first time that neuronal 620 inhibition of NF-κB signaling by expression of IκB-SR was able to mitigate the cytoplasmic 621 TDP-43 proteinopathy and behavioral deficits in transgenic models of ALS/FTLD 622 overexpressing TDP-43A315T or TDP-43G348C mutants. 623 624 In a previous study, we reported an interaction between TDP-43 and p65-NF-κB in the spinal 625 cord from ALS cases and from TDP-43 transgenic mice (Swarup et al., 2011b). Our results 626 suggested that such abnormal TDP-43/p65 interaction in ALS can enhance NF-κB activity. 627 Interestingly, the pro-oncoprotein Fused in Sarcoma (FUS), another RNA binding protein, was 628 also shown to bind p65-NF-κB and to serve as its co-activator(Uranishi et al., 2001).These 629 findings led us to propose that NF-κB signalling in neurons may constitute a therapeutic target 630 for ALS disease (Swarup et al., 2011b). So, to address the role of neuronal NF-κB activity, we 631 generated a transgenic mouse line expressingIκB-SRunder the control of neurofilament Nefh 632 gene promoter. After crossing the IκB-SR mice with TDP-43A315T and TDP-43G348C mice, the 633 resulting double transgenic mice exhibited as expected a reduced neuronal activation of NF-κB 634 activity as compared to single transgenic TDP-43A315T and TDP-43G348C mice. Thus, the levels of 635 phospo-p65-NF-κB immunostaining were significantly lower in nuclei of spinal neurons from 636 the double transgenic mice co-expressing IκB-SR when compared to single transgenic littermates 637 TDP-43A315T or TDP-43G348C (Fig. 2). Moreover, inhibition of NF-κB signaling by IκB-SR in 638 doubly transgenics resulted in a loss of interaction of TDP-43 with p65 NF-κB unlike the co- 639 immunoprecipitation of TDP-43 with p65 NF-κB in spinal cord extracts of TDP-43G348C mice 640 (Fig. 2H). 641 A remarkable outcome of neuronal inhibition of NF-κB activity by expression of IκB-SR in 642 TDP-43A315T or TDP-43G348C mice was the substantial reduction of cytoplasmic to nuclear ratio 643 of human TDP-43 as well as decrease in levels of aggregated TDP-43 recovered in the insoluble 644 fraction of spinal cord extract (Fig. 3). What mechanisms could explain such rescue of TDP-43 645 proteinopathy by inhibition of NF-κB activity? In normal conditions, TDP-43 shuttles between 646 the nucleus and cytoplasm mediating physiological functions at either locations (Ayala et al., 647 2008; Guo and Shorter, 2017). However, structural modifications may cause the protein to 648 accumulate in the cytoplasm with ensuing neurotoxicity(Barmada et al., 2010). Alterations at the

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649 RNA recognition motif 1 (RRM1) domain of TDP-43 by oxidation can induce its aggregation 650 and mislocalization in the cytoplasm (Chang et al., 2013; Shodai et al., 2013; Garnier et al., 651 2017). Moreover, normal RNA binding to this domain is necessary to prevent TDP-43 652 misfolding, oligomerization and subsequent accumulation (Zacco et al., 2018). There is 653 possibility that abnormal binding of p65NF-κB to the RRM1 domain of TDP-43 could interfere 654 with normal protein folding or with RNA binding, resulting in TDP-43 aggregation in the 655 cytoplasm. Accordingly, NF-κB inhibition would restore in part normal TDP-43 interactions. 656 This view is supported by our recent report that single chain antibodies targeting the RRM1 657 domain blocked interaction of TDP-43 with p65NF-κB and they restored normal TDP-43 658 functions (Pozzi et al., 2019). Another likely mechanism by which NF-κB inhibition may 659 contribute to the clearance of excess TDP-43 levels in the neuronal cytoplasm is by the induction 660 of protein degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. 661 Studies have shown that soluble form of cytosolic TDP-43 is primarily targeted for degradation 662 by the UPS whereas insoluble oligomeric forms tend to be cleared by autophagy (Scotter et al., 663 2014). NF-κB inhibition can lead to activation of the c-Jun N-terminal kinase (JNK) pathway 664 that ultimately facilitates the dissociation of Beclin1 from Bcl-2 thereby promoting autophagy 665 (Djavaheri-Mergny et al., 2006; Gao et al., 2016). Consistent with this view, in the doubly 666 transgenic TDP-43 mice co-expressing IκB-SR mice, there was an upregulation of autophagy 667 markers Beclin1 and LC3b which could possibly explain aclearance of cytoplasmic TDP-43 668 accumulations in spinal neurons. 669 670 The mitigation of TDP-43 proteinopathy by neuronal NF-κB inhibition in the double transgenic 671 mice was associated with a partial rescue of large spinal motor neuronsat one year of age and 672 with improved motor performance (Fig. 4). Furthermore, the neuronal expression of IκB-SR 673 ameliorated the cognitive deficits of mice expressing TDP-43 mutations as determined by the 674 novel object recognition assay and passive avoidance test (Fig. 5). These results are in line with 675 a previous report that conditional suppression of mutant TDP-43 in a transgenic mouse model 676 resulted in amelioration of cognitive performance (Ke et al., 2015). Cognitive decline during 677 aging is a characteristic feature of transgenic mice expressing mutant TDP-43 mice which is 678 reminiscent of human FTLD (Swarup et al., 2011a). 679

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680 We tested the effects of neuronal IκB-SR expression in the SOD1G93A mouse model which 681 develop a severe form of ALS-like disease with accumulation of misfolded SOD1 and massive 682 death of spinal motor neurons (Dion et al., 2009). To date, studies on the role of NF-κB in the 683 SOD1G93A model have been focused primarily to glial responses (Li et al., 2011; Kawabe et al., 684 2014; Ouali Alami et al., 2018; Schiaffino et al., 2018). Although inhibition of NF-κB activation 685 in astrocytes did not confer neuroprotection in mutant SOD1 mice (Crosio et al., 2011), 686 suppression of NF-κB in microglia extended life span and delayed onset of disease phenotypes 687 (Frakes et al., 2014). Here, our results revealed that selective suppression of neuronal NF-κB 688 activation in SOD1G93A caused a significant extension in their life expectancy (Fig. 8A). At all 689 stages of disease, the levels of misfolded SOD1 species were reduced supporting again the view 690 that neuronal NF-κB suppression may induce neuroprotection by clearance of excess misfolded 691 proteins via induction of autophagy. Thus, at 125 days of age, there was significantly more motor 692 neurons and ChAT reactivity in the spinal cord of double transgenic IκB-SR;SOD1G93Amice 693 when compared to SOD1G93A mice (Fig. 9). 694 In conclusion, our results suggest a key role for neuronal NF-κB activity in ALS pathogenesis 695 associated with TDP-43 proteinopathy or SOD1misfolding.It is widely acknowledged that 696 deregulated NF-κB signalling may contribute to acute neurodegenerative conditions such as 697 cerebral and traumatic brain injury(Harari and Liao, 2010) as well as chronic 698 neurodegenerative disorders such as Alzheimer’s disease (Jones and Kounatidis, 2017), 699 Parkinson’s disease (Flood et al., 2011), Huntington’s disease (Bečanović et al., 2015)and ALS 700 (Sako et al., 2012). However, an involvement of NF-κB activity in diseases is commonly 701 referring to pathogenic pathways of immune cells or glial cells. Our finding that suppression of 702 neuronal NF-κB activity with IκB-SR alleviated proteinopathies and behavioral phenotypes in 703 three mouse models of ALS or FTLD suggests that the NF-κB pathway in neurons constitutes a 704 promising therapeutic target. Thus, preclinical studies with ALS models are needed to test 705 pharmacological inhibitors of the neuronal NF-κB signalling pathway for their potency to confer 706 neuroprotection and, especially to mitigate TDP-43 pathology. 707 708 Author contributions 709 K.D. was involved in study design, performed experiments, analyzed data, prepared the figures 710 and wrote the manuscript; S.S.T. was the blinded observer for animal behavioral studies and

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711 performed statistical analysis; H.B. was involved in the generation of the Nefh-NF-κB1A (IκB- 712 SR) mice; C.B. genotyped the transgenic mice and maintained animal colonies; J.-P.J. was 713 involved in conceptualization and design of the study and manuscript preparation. 714 715 References 716 717 Arqués O, Chicote I, Tenbaum S, Puig I, G. Palmer H (2012) Standardized Relative 718 Quantification of Immunofluorescence Tissue Staining. Protocol Exchange. 719 Ayala YM, Zago P, D'Ambrogio A, Xu YF, Petrucelli L, Buratti E, Baralle FE (2008) Structural 720 determinants of the cellular localization and shuttling of TDP-43. Journal of cell science 721 121:3778-3785. 722 Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S (2010) Cytoplasmic 723 Mislocalization of TDP-43 Is Toxic to Neurons and Enhanced by a Mutation Associated 724 with Familial Amyotrophic Lateral Sclerosis. The Journal of Neuroscience 30:639-649. 725 Bečanović K et al. (2015) A SNP in the HTT promoter alters NF-κB binding and is a 726 bidirectional genetic modifier of Huntington disease. Nature Neuroscience 18:807. 727 Chang C-k, Chiang M-h, Toh EK-W, Chang C-F, Huang T-h (2013) Molecular mechanism of 728 oxidation-induced TDP-43 RRM1 aggregation and loss of function. FEBS Letters 729 587:575-582. 730 Conradi S, Ronnevi LO (1993) Selective vulnerability of alpha motor neurons in ALS: relation 731 to autoantibodies toward acetylcholinesterase (AChE) in ALS patients. Brain research 732 bulletin 30:369-371. 733 Crosio C, Valle C, Casciati A, Iaccarino C, Carrì MT (2011) Astroglial Inhibition of NF-κB 734 Does Not Ameliorate Disease Onset and Progression in a Mouse Model for Amyotrophic 735 Lateral Sclerosis (ALS). PLOS ONE 6:e17187. 736 Dion PA, Daoud H, Rouleau GA (2009) Genetics of motor neuron disorders: new insights into 737 pathogenic mechanisms. Nature Reviews Genetics 10:769. 738 Djavaheri-Mergny M, Amelotti M, Mathieu J, Besançon F, Bauvy C, Souquère S, Pierron G, 739 Codogno P (2006) NF-κB Activation Represses Tumor Necrosis Factor-α-induced 740 Autophagy. Journal of Biological Chemistry 281:30373-30382.

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771 Gusel'nikova VV, Korzhevskiy DE (2015) NeuN As a Neuronal Nuclear Antigen and Neuron 772 Differentiation Marker. Acta Naturae 7:42-47. 773 Harari OA, Liao JK (2010) NF-κB and innate immunity in ischemic stroke. Annals of the New 774 York Academy of Sciences 1207:32-40. 775 Hergesheimer RC, Chami AA, de Assis DR, Vourc'h P, Andres CR, Corcia P, Lanznaster D, 776 Blasco H (2019) The debated toxic role of aggregated TDP-43 in amyotrophic lateral 777 sclerosis: a resolution in sight? Brain : a journal of neurology 142:1176-1194. 778 Jones SV, Kounatidis I (2017) Nuclear Factor-Kappa B and Alzheimer Disease, Unifying 779 Genetic and Environmental Risk Factors from Cell to Humans. Frontiers in immunology 780 8:1805-1805. 781 Kawabe K, Kano O, Yanagihashi M, Ikeda K, Iwasaki Y (2014) Angiotensin-(1-7) inhibits NF- 782 kappa B in cells of ALS model mice. Journal of Neuroimmunology 275:118. 783 Ke YD, van Hummel A, Stevens CH, Gladbach A, Ippati S, Bi M, Lee WS, Kruger S, van der 784 Hoven J, Volkerling A, Bongers A, Halliday G, Haass NK, Kiernan M, Delerue F, Ittner 785 LM (2015) Short-term suppression of A315T mutant human TDP-43 expression 786 improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP 787 and ALS. Acta Neuropathol 130:661-678. 788 Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends in 789 neurosciences 19:312-318. 790 Lalancette-Hebert M, Sharma A, Lyashchenko AK, Shneider NA (2016) Gamma motor neurons 791 survive and exacerbate alpha motor neuron degeneration in ALS. Proceedings of the 792 National Academy of Sciences of the United States of America 113:E8316-E8325. 793 Li Q, Spencer NY, Pantazis NJ, Engelhardt JF (2011) Aand SOD1G93A regulate endosomal 794 ROS production by glial cells and pro-inflammatory pathways responsible for 795 neurotoxicity. Journal of Biological Chemistry. 796 Nelson PT et al. (2019) Limbic-predominant age-related TDP-43 encephalopathy (LATE): 797 consensus working group report. Brain. 798 Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck 799 T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, 800 Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM-Y (2006)

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801 Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral 802 Sclerosis. Science 314:130-133. 803 Ögren SO, Stiedl O (2010) Passive Avoidance. In: Encyclopedia of Psychopharmacology 804 (Stolerman IP, ed), pp 960-967. Berlin, Heidelberg: Springer Berlin Heidelberg. 805 Ohta Y, Tremblay C, Schneider JA, Bennett DA, Calon F, Julien J-P (2014) Interaction of 806 transactive response DNA binding protein 43 with nuclear factor κB in mild cognitive 807 impairment with episodic memory deficits. Acta neuropathologica communications 2:37- 808 37. 809 Ouali Alami N et al. (2018) NF‐κB activation in astrocytes drives a stage‐specific beneficial 810 neuroimmunological response in ALS. The EMBO Journal 37:e98697. 811 Pasetto L, Pozzi S, Castelnovo M, Basso M, Estevez AG, Fumagalli S, De Simoni MG, 812 Castellaneta V, Bigini P, Restelli E, Chiesa R, Trojsi F, Monsurrò MR, Callea L, 813 Malešević M, Fischer G, Freschi M, Tortarolo M, Bendotti C, Bonetto V (2017) 814 Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival 815 in a Mouse Model of Amyotrophic Lateral Sclerosis. The Journal of Neuroscience 816 37:1413. 817 Picher-Martel V, Dutta K, Phaneuf D, Sobue G, Julien J-P (2015) Ubiquilin-2 drives NF-κB 818 activity and cytosolic TDP-43 aggregation in neuronal cells. Molecular Brain 8:71. 819 Pozzi S, Thammisetty SS, Codron P, Rahimian R, Plourde KV, Soucy G, Bareil C, Phaneuf D, 820 Kriz J, Gravel C, Julien J-P (2019) Virus-mediated delivery of antibody targeting TAR 821 DNA-binding protein-43 mitigates associated neuropathology. The Journal of Clinical 822 Investigation 129. 823 Sako W, Ito H, Yoshida M, Koizumi H, Kamada M, Fujita K, Hashizume Y, Izumi Y, Kaji R 824 (2012) Nuclear factor kappa B expression in patients with sporadic amyotrophic lateral 825 sclerosis and hereditary amyotrophic lateral sclerosis with optineurin mutations. Clinical 826 neuropathology 31:418-423. 827 Schiaffino L, Bonafede R, Scambi I, Parrella E, Pizzi M, Mariotti R (2018) Acetylation state of 828 RelA modulated by epigenetic drugs prolongs survival and induces a neuroprotective 829 effect on ALS murine model. Scientific Reports 8:12875. 830 Scotter EL, Chen HJ, Shaw CE (2015) TDP-43 Proteinopathy and ALS: Insights into Disease 831 Mechanisms and Therapeutic Targets. Neurotherapeutics 12:352-363.

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832 Scotter EL, Vance C, Nishimura AL, Lee YB, Chen HJ, Urwin H, Sardone V, Mitchell JC, 833 Rogelj B, Rubinsztein DC, Shaw CE (2014) Differential roles of the ubiquitin 834 proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 835 species. Journal of cell science 127:1263-1278. 836 Shneider NA, Brown MN, Smith CA, Pickel J, Alvarez FJ (2009) Gamma motor neurons express 837 distinct genetic markers at birth and require -derived GDNF for postnatal 838 survival. Neural Dev 4:42. 839 Shodai A, Morimura T, Ido A, Uchida T, Ayaki T, Takahashi R, Kitazawa S, Suzuki S, Shirouzu 840 M, Kigawa T, Muto Y, Yokoyama S, Takahashi R, Kitahara R, Ito H, Fujiwara N, 841 Urushitani M (2013) Aberrant Assembly of RNA Recognition Motif 1 Links to 842 Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43). Journal of 843 Biological Chemistry 288:14886-14905. 844 Sun S, Elwood J, Greene WC (1996) Both amino- and carboxyl-terminal sequences within I 845 kappa B alpha regulate its inducible degradation. Mol Cell Biol 16:1058-1065. 846 Swarup V, Phaneuf D, Bareil C, Robertson J, Rouleau GA, Kriz J, Julien JP (2011a) 847 Pathological hallmarks of amyotrophic lateral sclerosis/frontotemporal lobar 848 degeneration in transgenic mice produced with TDP-43 genomic fragments. Brain 849 134:2610-2626. 850 Swarup V, Phaneuf D, Dupre N, Petri S, Strong M, Kriz J, Julien JP (2011b) Deregulation of 851 TDP-43 in amyotrophic lateral sclerosis triggers nuclear factor kappaB-mediated 852 pathogenic pathways. J Exp Med 208:2429-2447. 853 Uranishi H, Tetsuka T, Yamashita M, Asamitsu K, Shimizu M, Itoh M, Okamoto T (2001) 854 Involvement of the pro-oncoprotein TLS (translocated in liposarcoma) in nuclear factor- 855 kappa B p65-mediated transcription as a coactivator. J Biol Chem 276:13395-13401. 856 Urushitani M, Sik A, Sakurai T, Nukina N, Takahashi R, Julien JP (2006) Chromogranin- 857 mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral 858 sclerosis. Nat Neurosci 9:108-118. 859 Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphorylation of NF-kappaB and IkappaB 860 proteins: implications in cancer and inflammation. Trends Biochem Sci 30:43-52.

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861 Young K, Morrison H (2018) Quantifying Microglia Morphology from Photomicrographs of 862 Immunohistochemistry Prepared Tissue Using ImageJ. Journal of visualized experiments 863 : JoVE. 864 Zacco E, Martin SR, Thorogate R, Pastore A (2018) The RNA-Recognition Motifs of TAR 865 DNA-Binding Protein 43 May Play a Role in the Aberrant Self-Assembly of the Protein. 866 Frontiers in molecular neuroscience 11:372. 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902

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903 Figure legends 904 905 Figure 1: Generation and characterization of the IκB-super repressor mice 906 The neurofilament H (NFH) promoter was amplified from mouse genomic DNA and subcloned 907 into the pBluescript KS+ backbone. The Flag-EGFp and IκB (SS32, 36AA) fragments were 908 cloned from pEGFP-N3 and PCMV4-3HA/ IκBα (SS32, 36AA) plasmids respectively and 909 inserted into the pBSKS-Nefh promoter plasmid (A). Oocyets that were microinjected with this 910 plasmid were implanted into B6C3F1 mouse to generate the Nefh-IκBSS32,36AA mouse (a.k.a the 911 IκB-super repressor [SR]mouse). The founders were backcrossed with C57BL/6Ncrl mice prior 912 to cross-breeding with ALS mouse models (viz TDP-43A315T, TDP-43G348C and SOD1G93A) (B). 913 Genotyping of IκB-SR mice was performed to check a 329 bp fragment from the EGFP gene 914 which was absent in non-transgenic littermates (C). Spinal cord lysates from littermate IκB-SR 915 and WT mice were immunoprecipitated with Flag-M2 antibody and probed with IκBα antibody. 916 Data is representative image of 3 separate experiments. Results clearly showed a prominent band 917 at approximately 63 kDa corresponding to the combined molecular weight of FLAG-EGFP and 918 IκBα (dotted arrow), which was absent in the WT. This band could not be detected when the 919 Flag-M2 antibody was replaced with normal mouse IgG (D). To determine that the transgene 920 was not expressed in other non-neuronal cell types, spinal cord sections were co-stained for Flag 921 and Iba-1 (microglia marker) or Flag and GFAP (astrocyte marker). Flag expression could not be 922 detected from either Iba-1+ve or GFAP+ve cells (E). 923 924 Figure 2: Phospho-P65-NF-κB expression in spinal cord neurons and NF-κB-TDP-43 925 interaction 926 To assess the level of inhibition of p65-NF-κB activation, achieved due to the super repression, 927 and compare them with that in ALS mice models, spinal cord sections were stained with neuron 928 specific NeuN antibody and phopho-p65 NF-κB antibody. Nuclei were stained with DAPI. 929 Single channel images from IκB-SR;TDP-43A315T (Ai-iii), TDP-43A315T (Bi-iii), IκB-SR;TDP- 930 43G348C (Ci-iii), TDP-43G348C (Di-iii), IκB-SR (Ei-iii) and WT (Fi-iii) mice, were analyzed 931 separately using FIJI in a three step process. First, three corresponding single channel images 932 (NeuN, p65-NF-κB, DAPI) of every field were opened and stacked together. At the second step, 933 neuronal boundaries were drawn (based on NeuN staining) using the free-hand selection tool and

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934 the area was measured. Only neurons with an area > 250 μm2 were considered for this study. The 935 nuclear boundary was also marked similarly (based on DAPI). Finally, the p65-NF-κB signal in 936 the area marked as nuclear was measured and corrected for background fluorescence. For 937 representational purposes, all channels in (F) are showing original boundary markings. Neurons 938 from at least 6 sections per mouse with 3 mice in each group were considered. Magnification = 939 20X. The highlighted section in each single channel images were cropped using Adobe 940 Photoshop CS5, enlarged 300-fold, merged and displayed in actual colors (Aiv, Biv, Civ, Div, 941 Eiv and Fiv); scale bar = 100 μm. The data were tabulated and the Mean ± SEM plotted. 942 Analysis of variance was performed using Kruskall Wallis test followed by comparison between 943 groups using Dunn’s post-test. The adjusted P values are denoted in the figure (G). The neuronal 944 nuclear phospho-p65 NF-κB expression was found to be significantly elevated in both the ALS 945 mice model as compared to IκB-SR and WT. Neurons in the spinal cord of double transgenic 946 mice (IκB-SR;TDP-43 mutants) showed marked reduction in presence of phospho-p65 NF-κB in 947 comparison to respective TDP-43 mutants only. To assess whether the inhibition of p65-NF-κB 948 release from the inhibitory kappa B complex affected it’s binding with TDP-43, spinal cord 949 lysates from double transgenic mice (IκB-SR;TDP-43 mutant) and its littermate TDP-43 mutant 950 and IκB-SR were immunoprecipitated with anti-P65-NF-κB antibody and probed with anti- 951 hTDP-43 antibody. Result showed a clear reduction in binding between the two proteins due to 952 IκBα super repression (dotted arrow). No interaction was expectedly observed in IκB-SR mice 953 which lacked the human TDP-43 expression. Figure is representative of 3 independent 954 experiments (H). 955 956 Figure 3: TDP-43 expression in spinal cord neurons 957 To evaluate the nuclear-cytoplasmic distribution of hTDP-43 in spinal cord neurons of IκB- 958 SR;TDP-43A315T, TDP-43A315T, IκB-SR;TDP-43G348C and TDP-43G348C mice, sections were 959 stained with anti-human TDP-43 and anti-NeuN antibodies; nuclei were stained with DAPI (A- 960 D). Magnification = 20X. Boundaries (cellular and nuclear) were outlined using FIJI and TDP- 961 43 signal intensity was measured only from neurons with area > 250 μm2. The nuclear signal 962 intensity value was subtracted from the signal intensity of whole cell to generate the cytoplasmic 963 signal intensity. The cytoplasmic:nuclear (C:N) signal intensity was calculated and tabulated. 964 Neurons from at least 6 sections per mouse with 3 mice in each group were considered. A higher

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965 C:N ratio indicated predominantly cytoplasmic localization of TDP-43. Analysis of variance was 966 performed using Kruskall Wallis test followed by comparison between groups using Dunn’s 967 post-test. The adjusted P values are denoted in the figure. Results showed that TDP-43 was 968 predominantly cytoplasmic in neurons in spinal cord of TDP-43 mutant mice. In comparison, 969 there was a significant reduction in the C:N ratio of TDP-43 in spinal neurons of the double 970 transgenic mice. The C:N did not significantly differ either between the two TDP-43 mutant 971 groups or the two double transgenic groups (E). The hTDP-43 C:N ratio in both IκB-SR;TDP- 972 43A315T and TDP-43A315T groups correlated with increasing neuronal soma size as calculated by 973 Spearman’s rank correlation test (95% C.I.; ρ for Area v/s C:N of IκB-SR;TDP-43A315T = 0.4520; 974 p<0.0001; ρ for Area v/s C:N of TDP-43A315T = 0.2697; p=0.0033) (F). The hTDP-43 C:N ratio 975 in IκB-SR;TDP-43G348C did not significantly correlate with the neuronal soma area even though 976 that in TDP-43G348C did correlate with area (ρ for Area v/s C:N of IκB-SR;TDP-43G348C = 977 0.1677; p=0.0575; ρ for Area v/s C:N of TDP-43G348C = 0.2519; p=0.0079) (G). Immunobloting 978 from RIPA soluble (H) and insoluble (I) fractions of spinal cords of mice from various groups 979 showed that in both IκB-SR;TDP-43A315T and IκB-SR;TDP-43G348C mice spinal cords, human 980 TDP-43 was present at a significantly higher level as compared to respective TDP-43 mutant 981 mice tissues (J). Contrarily, in the RIPA insoluble fractions, more human TDP-43 was detected 982 from the TDP-43 mutant mice tissues (K). Data was analyzed by unpaired Student’s T test with 983 Welch’s correction. P values are denoted in the figure. 984 985 Figure 4: Motor activities of the transgenic mice 986 987 To assess the effect of NF-κB activation inhibition on motor neuron count in IκB-SR;TDP- 988 43A315T and IκB-SR;TDP-43G348C mice and compare them with that in age-matched TDP- 989 43A315T, TDP-43G348C, IκB-SR and WT littermates, spinal cord sections were stained for the 990 neuronal marker NeuN (A). Magnification 20X; scale = 100 μm. The number of NeuN positive 991 cells in ventral horn with an area > 250 μm2 were counted using FIJI and data was represented as 992 a box-whisker graph showing minimum to maximum range of values. The data was analyzed by 993 one-way ANOVA with Tukey’s post-test that clearly showed that 1 year old IκB-SR;TDP-43 994 mutant mice had significantly more motor neurons in comparison to respective TDP-43 mutants. 995 However, these levels were still not at par with those observed in IκB-SR or WT. Data is

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996 representative of 3-5 mice per group. The adjusted P values are denoted in the figure (B). To 997 determine if the spinal motor neuron count translated to motor performance, the mice in all six 998 groups were trained to run on an accelerating rotarod from 4 months of age. Data was recorded 999 weekly, till 12 months of age and represented as the mean of maximum latency to fall from the 1000 rotarod at each time point. N=10-15 per group (C). Comparison of rotarod performance of WT 1001 and IκB-SR mice does not show any significant difference over the duration of the protocol (4 to 1002 12 months) (D). Comparison of motor performances of mice belonging to IκB-SR;TDP-43A315T 1003 and TDP-43A315T groups reveled an initiation of decline in the TDP-43 mutant mice at about 280 1004 days of age. However, owing to the variance in results, this became statistically significant 1005 difference beyond 330 days of age. In contrary, the IκB-SR;TDP-43A315T mice maintained their 1006 performance at a steady level. Data was analyzed by multiple T test- one per row (E). Similar 1007 pattern of decline was observed while comparing between IκB-SR;TDP-43G348C and TDP- 1008 43G348C. However, in TDP-43G348C mice, the decline was initiated at an earlier time point (225 1009 days) and their motor performance at this age was significantly different from that of IκB- 1010 SR;TDP-43G348C mice (F). Data was analyzed by multiple T test- one per row and the P values 1011 are denoted in the figure. 1012 1013 Figure 5: Determination of cognitive abilities in TDP-43 mutant and IκB-super repressor 1014 mice 1015 Novel object recognition test performed at 6 months (A) and 12 months (B) of age showed mice 1016 expressing mutant TDP-43 with impaired recognition of the novel object. Contrarily, IκB- 1017 SR;TDP-43 mutant mice performed relatively similar to wild type or IκB-SR mice. The data is 1018 representative of independent mice tested (dots) and represented as Mean ± SEM of percent time 1019 spent at the novel object. Data were analyzed using one way ANOVA with Tukey’s multiple 1020 comparison post-test. The adjusted P values are denoted in the figure. Passive avoidance test to 1021 evaluate fear-induced memory retention indicated cognitive decline in TDP-43G348C mice at 12 1022 month of age, in comparison to littermate WT and IκB-SR mice. The IκB-SR;TDP-43G348C 1023 double transgenic mice performed significantly better in the memory function test in comparison 1024 to the TDP-43G348C mice. No significant difference was observed between performance of IκB- 1025 SR;TDP-43A315T and TDP-43A315T mice, which in either cases was significantly less than 1026 littermate WT or IκB-SR mice. The data is representative of independent mice tested (dots) and

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1027 represented as Mean ± SEM of time (in seconds) taken to enter the dark chamber. Data were 1028 analyzed using one way ANOVA with Tukey’s multiple comparison post-test. The adjusted P 1029 values are denoted in the figure (C). Immunoblot analysis from hippocampal extracts (RIPA 1030 soluble and insoluble fractions) of 3 non-littermate TDP-43A315T and TDP-43G348C mice showed 1031 significantly higher TDP-43 expression in TDP-43A315T. Data was analyzed by 2 way ANOVA 1032 followed by Tukey’s multiple comparison test. The adjusted P values are denoted in the figure 1033 (D). 1034 1035 Figure 6: Effect of IκB-super repression on glial activation pattern in spinal cord 1036 Spinal cord sections stained with anti-GFAP antibody to visualize astrocytes in the six groups; 1037 Magnification = 40X, scale bar 50 μm. Images are representative of 3 mice per group (A). The 1038 GFAP signal intensity from ventral horn was quantified using FIJI and data represented in 1039 tabular form. Results show that GFAP signal intensity to be significantly elevated in spinal cords 1040 of TDP-43 mutant mice that could indicate astrogliosis and/or reactive astrocytosis. The signal 1041 intensities were significantly lesser in IκB-SR;TDP-43 mutant mice. Data is representative of at 1042 least 6 section each from 3 mice per group. Data was analyzed by one way ANOVA along with 1043 Tukey’s multiple comparison post-test. The adjusted P values are denoted in the figure (B). To 1044 visualize microglial morphology, spinal cord sections were stained with anti-Iba1 antibody. 1045 Images are representative of 3 mice per group; Magnification = 20X, scale bar 50 μm (C). The 1046 Iba-1 signal intensity from ventral horn was quantified using FIJI and data represented in tabular 1047 form. Data shows signal intensity to be significantly higher in spinal cords of TDP-43 mutant 1048 mice and also IκB-SR mice as compared to IκB-SR;TDP-43 mutants or WT mice. Data is 1049 representative of at least 6 section each from 3 mice per group. Data was analyzed by one way 1050 ANOVA along with Tukey’s multiple comparison post-test. The adjusted P values are denoted in 1051 the figure (D). Spatiotemporal analysis of microglial morphology was done also with the help of 1052 FIJI. The cells were converted to their skeleton form (E) followed by analysis of number of 1053 branches (processes) per cell (F) and total branch (process) length of each cell (G). Results show 1054 that microglia in spinal cord of TDP-43 mutant mice have significantly reduced number of 1055 branches as well as decreased total branch length as compared to IκB-SR;TDP-43 mutants or 1056 IκB-SR or WT mice implying a more amoeboid reactive morphology. Each dot represents data 1057 from individual neurons. Data is pooled from at least 6 sections each from 3 mice per group.

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1058 Data was analyzed by one way ANOVA along with Tukey’s multiple comparison post-test. The 1059 adjusted P values are denoted in the figure 1060 1061 Figure 7: Changes in autophagy markers due to IκB-super repression 1062 Spinal cord sections were stained for different markers of autophagy- LC3B, P62 and ATG5. 1063 Images in the figure are representative of 3 mic per group. Magnification = 20X; Scale bar = 50 1064 μm (A and B). The signal intensity of these markers in spinal cord neurons with an area > 250 1065 μm2 were measured using FIJI and the data is represented in tabular form (C, D and E). Results 1066 clearly shows significantly reduced expression of autophagy markers in spinal cord neurons of 1067 TDP-43 mutant mice compared to IκB-SR;TDP-43 mutants or WT mice. The levels of P62 and 1068 ATG5 were also found to be significantly higher in IκB-SR mice compared to WT. Immunoblot 1069 performed from spinal cord lysates to detect Beclin showed an increase in the protein expression 1070 in IκB-SR mice as compared to WT and TDP-43 mutants. The Beclin expression in IκB- 1071 SR;TDP-43 mutant mice were slightly elevated in comparison to only TDP-43 mutants. Data is 1072 representative of 3 mice per group. Data was analyzed by One way ANOVA with Tukey’s 1073 multiple comparison post-test. The adjusted P values are denoted in the figure (F-G). 1074 1075 Figure 8: IκB-super repression extends life span of SOD1G93A mice and slows rate of disease 1076 progression 1077 On comparing lifespan of IκB-SR;SOD1G93A mice with that of only SOD1G93A, a median 1078 increase of 15 days was observed. Data was analyzed by Mantel-Cox’s Log rank test; n= 25 in 1079 each group; p= 0.0020 (A). Rotarod analysis clearly showed a decline in performance by IκB- 1080 SR;SOD1G93A and SOD1G93A relative to that in IκB-SR and WT mice. The performance of 1081 SOD1G93A mice was found to differ significantly from that in IκB-SR;SOD1G93A between 133 1082 and 140 days of age. Data was compared by Student’s Test along with Welch’s correction at 1083 each time point; the P values are denoted in the figure. N= 25 in IκB-SR;SOD1G93A; 15 in 1084 SOD1G93A; 8 in IκB-SR and 10 in WT (B). The grid hang time of the IκB-SR;SOD1G93A and 1085 SOD1G93A mice were also clearly worse than IκB-SR and WT mice. However, no significant 1086 difference were observed at any time point between IκB-SR;SOD1G93A and SOD1G93A mice; n= 1087 25 in IκB-SR;SOD1G93A; 15 in SOD1G93A; 8 in IκB-SR and 10 in WT (C). Hind limb reflex 1088 scoring showed a slower rate of decline in IκB-SR;SOD1G93A mice in comparison to SOD1G93A

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1089 between 141 and 161 days of age (denoted by dotted lines). Data was compared by Student’s 1090 Test along with Welch’s correction at each time point and the P values are listed in Table 1-1; 1091 n= 20 in IκB-SR;SOD1G93A; 15 in SOD1G93A (D). The actual body weight comparison did not 1092 show any difference between IκB-SR;SOD1G93A and SOD1G93A over their life span (E). 1093 However, on converting the body weights as percentage of peak weight, a significant difference 1094 could be observed between 126 and 147 days of age. Data was compared by Student’s Test along 1095 with Welch’s correction at each time point; the P values are denoted in the figure; n= 20 in IκB- 1096 SR;SOD1G93A; 15 in SOD1G93A (F). 1097 1098 Figure 9: Neuronal IκB-super repression prevents mis-folding of SOD1 and extends motor 1099 neuron survival in SOD1G93A model 1100 Levels of mis-folded SOD1 was detected in spinal cord lysates at pre-symptomatic, early 1101 symptomatic and at end stage by immunoprecipitation. In comparison to SOD1G93A, IκB- 1102 SR;SOD1G93A always presented lesser amount of mis-folded SOD1. Data is representative of 3 1103 mice per group (A). Quantitative analysis of the immunoblots also reflects this tread. However, 1104 the difference in the levels of mis-folded SOD1 between IκB-SR;SOD1G93A and SOD1G93A were 1105 not statistically significant (B). Spinal cord sections from both IκB-SR;SOD1G93A and SOD1G93A 1106 mice were immunostained to detect ChAT. ChAT expression was found to be pronounced in 1107 spinal cord of IκB-SR;SOD1G93A mice in comparison to only SOD1G93A mice. Magnification= 1108 20X; scale bar= 50 μm. Data is representative of 3 mice per group (C). Motor neuron numbers in 1109 spinal cord sections were estimated by staining them with NeuN and counting positively stained 1110 cells with an area >250 μm2. Images are representative of 3 mice per group. Magnification= 1111 20X; scale bar= 50 μm (D). The data were tabulated and represented as box-whisker graph 1112 showing minimum to maximum range of values. The data was analyzed by one-way ANOVA 1113 with Tukey’s post-test; the adjusted P values are denoted in the figure (E). 1114 1115 Figure 1-1: Characterization of the IκB-SR mice 1116 Spinal cord sections from the 4 founder mice lines, along with non-transgenic littermate, were 1117 sectioned and stained with anti-FLAG antibody. Strongest signal was detected from founder line 1118 1397, whose sections were further stained with anti-GFP antibody to confirm presence of 1119 transgene. Magnification=20X; scale bar= 100 μm (A). Spinal cord lysates were also

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1120 immunoblotted and probed with anti-FLAG and anti-GFP antibodies. The protein expressed by 1121 the transgene (approximately 63 kDa; denoted by dotted arrow) could be detected in some 1122 founders but never in WT (non-transgenic) mice (B). Similar results were obtained from brain 1123 lysates. Data is representative of at least 3 mice per group (C). Kidney, liver and spleen from 3 1124 littermate mice from founder line 1397 were lysed and probed for FLAG and GFP. Lack of any 1125 specific signal confirmed that the expression of transgene was limited to (D). IκB- 1126 SR and WT mice were intraperitoneally challenged with E. coli LPS to induce inflammation and 1127 sacrificed after 12h. Spinal cord and spleen were excised and lysed for immunoblotting. When 1128 probed for phospho IκBα and total IκBα, transgenic mice showed no induction in spinal cord but 1129 robust induction in spleen. However, IκBα was found to be induced in both spinal cord as well as 1130 spleen in WT mice (E). 1131 1132 Figure 2-2: Miscellaneous data 1133 The IκB-SR plasmid was cloned into a pcDNA3 backbone. Mouse motor neuron-like NSC34 1134 cells were transfected with this plasmid, with or without exposing the cells to recombinant 1135 murine TNF-α (10ng/mL). The levels of phospho NF-κB and total NF-κB were estimated from 1136 the cell lysates. In un-transfected cells, TNF-α treatment resulted in increased phosphorylation of 1137 NF-κB which was not observed in case of cells that were transfected with the IκB-SR plasmid 1138 (A). A 40X magnified image of spinal cord neuron, stained for NeuN and hTDP-43 with borders 1139 (cellular and nuclear) drawn around it prior to measurement of nuclear and cytosolic signal 1140 intensities (B). Body weights of all male and female mice from the six groups showing no 1141 significant intergroup variation which could affect performance in motor performance test (C). 1142 1143 1144 1145 1146

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Table 1. Details of antibodies used for immunobloting (IB), immunoprecipitation (IP) and immunofluorescence (IF).

Antibody against Dilution for IB/IP Dilution for IF Company Millipore, Temecula, CA, Actin 1:15000 USA Novus Biologicals; Littleton, Atg-5 1:250 CO, USA Novus Biologicals; Littleton, Beclin-1 1:1000 CO, USA Millipore, Temecula, CA, ChAT 1:500 USA 1:2500 Sigma-Aldrich, St. Louis, FLAG-M2 *2 μg/300 μg of total 20 μg/mL MO, USA protein Santa Cruz, Dallas, TX, GAPDH 1:2500 USA Cell Signaling Technologies, GFAP 1:300 Beverly, MA, USA Thermo Fischer, Rockford, GFP 1:1000 1:300 IL, USA. Wako Chemicals, Iba-1 1:500 Richmond, VA, USA Santa Cruz, Dallas, TX, IkBα 1:2000 USA Cell Signaling Technologies, Phospho-IkBα 1:1000 Beverly, MA, USA Novus Biologicals, LC3-II 1:250 Littleton,CO, USA Cell Signaling Technologies, NeuN 1:500 Beverly, MA, USA 1:1000 Santa Cruz, Dallas, TX, NFκB *1.5μg/300 μg of total USA protein Phospho-NFκB (Ser Cell Signaling Technologies, 1:1000 536) Beverly, MA, USA SOD1 mAb *1 μg/300 μg total Generated from hybridoma (misfolded; clone protein in the lab. B8H10) Enzo Life Sciences; SOD1 pAb 1:5000 Farmingdale, NY, USA. Proteintech; Rosemont, IL, Pan TDP-43 1:2500 USA. Thermo Fischer, Rockford, Human TDP-43 1:1000 IL, USA.

1

Abnova; Taipei City, Human TDP-43 1:250 Taiwan.

*For immune-precipitation

2