Deletion of Ripk3 Prevents Motor Neuron Death In Vitro but not In Vivo

Abstract Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in both in vitro and in vivo models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors Ripk1, Ripk3, and Mlkl in the spinal cord of mutant superoxide dismutase-1 (SOD1G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1G93A mice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing of Ripk3 in cultured MNs protected them from toxicity associated with SOD1G93A astrocytes. However, constitutive deletion of Ripk3 in Tg SOD1G93A mice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit of Ripk3 silencing in vivo. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1G93A or Optineurin knock-out mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1G93A-induced neurodegeneration and call for further preclinical investigations to determine if necroptosis plays a critical role in the pathogenesis of ALS.


Introduction
ALS is an adult-onset paralytic disorder that is characterized by the loss of upper and lower motor neurons (MNs). Neurodegeneration results in muscle weakness leading to difficulty in moving, speaking, swallowing and eventually breathing. The disease progresses rapidly with a mean survival of three to five years after diagnosis. To date, riluzole and edaravone are the only two Food and Drug Administration-approved ALS drugs, each of which has only marginal therapeutic benefit (Miller et al., 2007;Abe et al., 2014). Given these facts, it is estimated that more than two in every 100,000 Americans will die from ALS, unless a more effective treatment is found. Advances in our knowledge of the mechanisms involved in disease pathogenesis will undoubtedly lead to the development of novel therapies for ALS.
Inhibition or deletion of the death agonist Bax prevents MN degeneration in both cell and animal models of ALS (Gould et al., 2006;Nagai et al., 2007;Re et al., 2014). In contrast, selective antagonists of executioner caspases and the pan-caspase inhibitor zVAD do not attenuate MN loss in cell culture models of familial and sporadic ALS (Nagai et al., 2007;Re et al., 2014) and are only mildly protective in Tg SOD1 G93A ALS mice . Furthermore, while some authors have shown evidence of caspase activation in ALS mouse models and even in human tissue (Friedlander et al., 1996;Martin, 1999;Pasinelli et al., 2000;Vukosavic et al., 2000;Inoue et al., 2003), others have failed to detect clear signs of apoptosis in degenerating MNs in mouse models of ALS (Migheli et al., 1999). Collectively, these findings argue that a caspase-independent form of programmed cell death (PCD) may underlie neurodegeneration in ALS. Previous studies focusing on the molecular basis of MN death in in vitro models (Nagai et al., 2007;Re et al., 2014;Ikiz et al., 2015) have demonstrated that pharmacological or genetic targeting of key determinants of necroptosis, a molecularly-controlled form of necrosis (Grootjans et al., 2017) prevented MN death (Re et al., 2014. Further evidence that necroptosis may be involved in ALS neurodegeneration comes from in vivo studies in which the administration of the small molecule Nec-1s, a kinase antagonist of the receptor-interacting protein kinase 1 (RIPK1), or deletion of RIPK3, two key determinants of necroptosis (Grootjans et al., 2017), was shown to delay the onset of the ALS phenotype in Tg SOD1 G93A mice (Ito et al., 2016). These authors (Ito et al., 2016) also showed that inhibiting necroptosis rescued axonal myelination defects in both Tg SOD1 G93A mice and in mice deficient in optineurin (Optn Ϫ/Ϫ ), another ALS mouse model.
Given the potential therapeutic value of targeting necroptosis in ALS, the goals of the present study were first, to confirm the involvement of this form of PCD in ALS neurodegeneration by quantifying transcript and protein levels of core necroptosis determinants in affected regions of the central nervous system. Second, since previous studies only reported on the effects of targeting necroptosis on the onset of motor dysfunctions (Ito et al., 2016), we sought to characterize the benefits of an antinecroptosis strategy on motor performance and survival in ALS mice to provide preclinical evidence to support this approach to treat ALS patients. Third, since Ito et al. (2016) invoke activation of necroptosis in both oligodendrocytes and microglia, but not in spinal MNs, while in vitro models suggest the activation of necroptosis within MNs, we aimed here to resolve this discrepancy by elucidating the cellular site of action of necroptosis in ALS.

Animals
All experimental procedures followed the National Institutes of Health Guide for Care and Use of Laboratory Animals (National Research Council, 2011). All animal procedures were approved and performed in accordance with the institutional animal care and use committee's policies at Columbia University. A total of ϳ75 male and female mice were group-housed in polycarbonate cages with corncob bedding; they were maintained in a humidity-and temperature-controlled vivarium (20 -22°C) on a 12/12 h light/dark schedule. Animals had access ad libitum to food and water except during behavioral testing.
Optn tm1a(EUCOMM)Wtsi ES cells were purchased from The Jackson Laboratories (http://www.informatics.jax.org/allele/MGI:4432769). Using standard protocol, ES cells were then injected into the blastocoel cavity of 3.5-d-old mouse blastocysts which were transferred surgically to the uterine horns of appropriately timed pseudo-pregnant recipient females which gestated normally (Conner, 2001). Chimeric pups were then genotyped and used for further crossing to generate Optn knock-out mice. Optn mutation details: the L1L2_Bact_P cassette was inserted at position 5053776 of chromosome 2 upstream of the critical exon(s) (Build GRCm38). The cassette was composed of a FRT site followed by a lacZ sequence and a loxP site. This first loxP site was followed by neomycin under the control of the human beta-actin promoter, SV40 poly A, a second FRT site and a second loxP site. A third loxP site was inserted downstream of the targeted exon at position 5052564. LoxP sites thus flanked the critical exon. A "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele. Subsequent UBC-cre expression resulted in a knock-out mouse. After one more cross, Optn knock-out homozygous mice were generated and used for the experiments; littermates were used as controls. For Optn, genotyping was performed using primer sets: forward: 5=-GCAGGGGCATTCTAAGTTCA-3=, reverse: 3'-TCCCTGCAAATTCCTTTCTG-5' and forward: 5=-T C T G A A C C C C A A A C A G A A G C -3 = , r e v e r s e : 5 = -GCTCTTCCTTCAGCCTCTCA-3=, for WT and knock-out Optn, respectively. Optn ϩ/ϩ (n ϭ 3) and Optn Ϫ/Ϫ (n ϭ 3) were assessed for MN number in the lumbar spinal cord and for innervation of the NMJ at the TA muscle.
For mixed-lineage kinase domain-like (Mlkl) silencing, L929 cells were infected with lentivirus-containing shRNA against Mlkl (360819; Sigma Mission) at a multiplicity of infection (MOI) 100. Four days later, cells were harvested and protein extraction was performed to assess knockdown efficiency.

RNA extraction-cDNA synthesis-qPCR
Total RNA was extracted from lumbar spinal cords (L1-L5 segment) using TRI reagent (T9424; Sigma) following the manufacturer's protocol. DNase treatment was performed using rDNAseI (AM2235; Ambion) to remove any remaining DNA, followed by phenol chloroform extraction to ensure high RNA quality. RNA concentration was determined spectrophotometrically at 260 nm. Quality of the RNA was determined by the 260/280 and 260/ 230 ratio. cDNA was generated with the RevertAid First Strand cDNA Synthesis kit (K1691; ThermoFisher) following manufacturer's protocol. For the reaction, we used 1 g of RNA primed with both random hexamers and oligo(dT) primers. A three-step real-time qPCR was conducted with the Realplex 4 Mastercycler PCR System (Eppendorf) using SYBR Green dye (4367659; Thermo-Fisher).

Lentiviral-mediated gene silencing in mouse PMN cultures
Primary neuronal cultures from E12.5 mouse embryos were prepared as described above and diluted at a final concentration of 1 ϫ 10 5 cells/ml. Next and before plating, cells were infected with pLKO.1-puro plasmids (Sigma Mission); TRCN0000022468 clone (targeting Ripk1); or the SHC002H (scrambled) at a MOI of 20 following the spinoculation protocol (https://www.sigmaaldrich.com/life-science/ functional-genomics-and-rnai/shrna/learning-center/ spinoculation-protocol.html). Cells were then centrifuged at 800 ϫ g for 30 min at room temperature, resuspended in fresh PMN media, and seeded at a density of 120,000 cells on 0.01% poly-D-lysine-coated (P1274; Sigma) and 15 g/ml laminin-coated (23017-015; Invitrogen) 24-well plates. Four days after infection, cells were harvested and assessed for RIPK1 protein expression by Western blotting. For MNs, infections were performed in the absence of hexadimethrine bromide and puromycin, since the addition of these factors did not enhance the knock-down efficiency in these nondividing cells.

Western blotting
Mice were anaesthetized with ketamine-xylazine and perfused intracardially with 0.1 M ice-cold PBS (4190136; Ther-moFisher) for 4 min at 10 ml/min. Spinal cords were removed, frozen on dry ice, and stored at -80°C in preweighed tubes. On the day of the lysis, cords were thawed on ice and weighed. Lysis buffer was added at a ratio of 1 ml/100 mg of tissue.
For RIPA lysis buffer, tissue was first homogenized then sonicated (2.5 Hz, 10 s, two times), and clear lysate was isolated following centrifugation at 13,000 rpm for 15 min. For the 6 M urea lysis buffer, tissue was first homogenized in buffer containing all of the ingredients except 6 M urea, then sonicated (as for RIPA) and incubated for 1 h at 4°C with rotation. After centrifugation at 15,000 rpm for 20 min, the supernatant was removed and the pellet was washed twice with PBS (14190136; ThermoFisher). A total of 6 M urea-containing buffer was added and the pellet was resuspended, sonicated, then re-incubated at 4°C with rotation for 1 h. Protein concentrations were determined using the Bradford Quick Start assay (500-0205; Bio-Rad) and DC Protein Assay (Bio-Rad) for the RIPA and the 6 M urea lysis buffer, respectively. Approximately 50 g of protein in the lysates was mixed with 5ϫ Laemmli buffer (Tris-HCl pH 6.8, 10% SDS, 25% glycerol, 5% ␤-mercaptoethanol, and 0.05% bromophenol blue) to a final concentration of 1ϫ before running on 4 -12% Bis-Tris precast gels (NP0341BOX; Life Technologies). Following transfer to a nitrocellulose membrane, the blots were probed with antibodies directed against: RIPK1 (1: 1000; AB_394014), RIPK3 (1:1000; AB_2722663), MLKL (1:1000; AB_11134649), p-MLKL (1:1000; AB_2687465) overnight at 4°C. ␤-ACTIN (1:40,000; AB_476744) and GAPDH (1:10,000; AB_1080976) were used as loading control. Blots were probed with either fluorescent (Li-Cor buffer 1:20,000 IR-700 dye conjugated; mouse, rabbit) or HRP-conjugated (mouse, rabbit) secondary antibodies (1: 3000; NXA931: mouse, NA93AV: rabbit, GE Healthcare UK Ltd). Fluorescent imaging was performed with the Li-Cor Odyssey Imaging system. Chemiluminescent imaging was performed with Supersignal West Pico Chemiluminescent Substrate (34080; ThermoScientific) and visualized with X-Ray Films (Medilink Imaging). Following scanning of the images, ImageJ analyzer was used to quantify the optical density of the bands. In all cases, the levels of the proteins of interest were normalized to those of ␤-ACTIN or GAPDH for quantification and statistical analysis.

Transmission electron microscopy (TEM)
TEM was performed as described previously (Sosunov et al., 2017). Briefly, mice were anaesthetized with isoflurane before intracardiac perfusion with ice-cold PBS followed by ice-cold 2.5% glutaraldehyde ϩ 2% PFA in 0.1 M phosphate buffer (pH 7.4). Spinal cords with ventral and dorsal roots at the lumbar level (L1-L4) were removed under a dissecting microscope and kept in the fixative for 12-16 h (4°C). After postfixation in 2% osmium tetroxide in 0.2 PBS (2 h at 4°C) and dehydration, small pieces of tissue were embedded in Epon-Araldite (Electron Microscopy Sciences). Semi-thin sections stained with toluidine blue were used for orientation. Ultrathin sections were cut with Reichert Ultracut E, contrasted with uranyl acetate and lead citrate, and examined with a JEOL 1200 electron microscope. For each genotype, three to four mice were used.
Negative Results Figure 1. Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1 G93A mice. Lumbar spinal cords, from 12-and 15-week-old Tg SOD1 G93A , WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, MLKL, and p-MLKL. A, Quantification of Ripk1, Ripk3, and Mlkl mRNA from 12-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1 G93A compared to Tg SOD1 WT (p ϭ 0.0021) and NTg (p ϭ 0.0076) at 12 weeks. B, Quantification of Ripk1, Ripk3, and Mlkl mRNA in spinal cords of 15-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk1 (p ϭ 0.0009, vs Tg SOD1 WT ; p ϭ 0.0020, vs NTg) and Ripk3 (p ϭ 0.0115; vs Tg SOD1 WT , p ϭ 0.0067; vs NTg) but not for Mlkl in Tg SOD1 G93A compared to Tg SOD1 WT and NTg mice at 15 weeks. C, Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1 WT , and Tg SOD1 G93A 15-week-old mice. ␤-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral shRNA in mouse PMN cultures (mPMNs). D, Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1 G93A samples compared to Tg SOD1 WT (p ϭ 0.0279) and NTg (p ϭ 0.0033) mice. Results are presented as mean Ϯ SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey's post hoc analysis; n ϭ 3 biological replicates per genotype. E, Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Non-specific band at 47 kDa is designated as an asterisk ‫.)ء(‬ NTg spleen: positive control tissue, Ripk3 Ϫ/Ϫ spleen and Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ spinal cord: negative control tissue. F, Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). Ripk3 Ϫ/Ϫ spinal cord: negative control tissue. G, Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H, Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1 G93A ). NIH 3T3: positive control cell lysate. I, Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.

Mouse behavioral tests
The loaded grid test was performed as previously described by Barnéoud et al. (1997). Briefly, the mouse was suspended by the tail and allowed to grip a series of grids with increasing weights (10, 20, 30, 40 g) and a behavioral score was calculated as follows: score ϭ [⌺(tw ϫ W)]/body weight, where tw corresponds to the maximum time the mouse was able to hold the grid at weight W. Body weight is the weight of the animal at the time of the test. A maximum period of 30 s was allowed for each weight. The best of three trials was recorded with at least 15-s resting period between each trial of the same weight and at least 30-s resting period between each new weight. Inverted grid or "wire-hang" test was performed by first allowing the mouse to grip a grid and then inverting the grid, thus allowing the mouse to hang. Score was recorded as maximum hold time up to 60 s, as the best of three trials with at least 3-min resting period between each trial. This test was always performed on a day other than the loaded grid test day. Animals were tested once per week at roughly the same time of day throughout the trial.

Quantification of innervated NMJs of the TA muscle
Mice were anaesthetized with ketamine-xylazine and perfused intracardially with 0.1 M ice-cold PBS (4190136; ThermoFisher) for 4 min at 10 ml/min followed by ice-cold 4% PFA in 0.1 M PBS for 8 min at 10 ml/min. TA muscle was dissected out and incubated in PFA overnight at 4°C, then transferred to 30% sucrose in 0.12 M phosphate buffer at 4°C for at least 48 h. The TA muscles were subsequently placed in OCT (Tissue-Tek) in molds on dry ice and stored at -80°C until cryosectioning. Cryosections of TA (20 m) were cut and slide-mounted. NMJs were immunolabeled for presynaptic marker VaChT (1:2000; AB_261875) or synaptophysin (Syn; 1: 500; AB_86671) or postsynaptic nicotinic acetylcholine receptors using fluorescent-conjugated BTX (1:200 ␣-bungarotoxin conjugated to Alexa Fluor 594; Invitrogen). NMJ innervation was quantified by identifying BTX-positive NMJs (red) and determining the extent of co-localization with VAChT (green). Full innervation is considered greater than a 70% overlap of BTX with VaChT, partial at 30 -70% overlap, and denervation at 0 -30% overlap. Imaging was performed with a fluorescence microscope at 10ϫ magnification. At least 100 NMJs on 20-m TA sections were imaged and counted for each mouse.

Quantification of MNs in the mouse spinal cord
Mice were anaesthetized with ketamine-xylazine and perfused intracardially as described above. Spinal cords were removed and postfixed in PFA overnight at 4°C. The L4-L5 segment, identified by its ventral roots, was carefully dissected out and incubated at 4°C in 10% sucrose in 0.12 M phosphate buffer for at least 48 h. The spinal cord was then incubated in 7.5% gelatin/10% sucrose solution at 37°C for 1.5 h followed by embedding in a gelatin/sucrose solution in a plastic mold at 4°C until firm for not Ͼ1 d. Spinal cords in gelatin were then cut into blocks and flash frozen for 45 s in 2-methylbutane, dry ice cooled to -60°C and stored at -80°C until sectioning; 20-m sections were cut on a cryostat and every other section was collected for immunostaining with the MN-specific marker ChAT (1:100; AB_2079751). An entire set of minimum 100 sections (20-m thickness) from L4-L5 was counted, and the average number of MNs per ventral horn was obtained. A neuron would be counted if the nucleus, as identified by a DAPI counterstain, was present in the optical plane.

Statistics
All datasets are expressed as mean Ϯ SEM. Differences between variables were analyzed by Student's t test (with Bonferroni correction to correct for multiple comparisons, whenever indicated) and among variables by one-way ANOVA or two-way ANOVA followed by Tukey's or Newman-Keuls post hoc tests, respectively. RIPK1 protein levels and clinical information were tested for correlation by linear regression. Survival and onset were analyzed via log-rank Mantel-Cox test. All statistical analyses were run on SigmaPlot 12.0 (Systat Software, Inc.), and the null hypothesis was rejected at the 0.05 level, unless corrected for multiple comparisons.

Results
Expression of key molecular determinants of necroptosis in Tg SOD1 G93A mice As a first step toward confirming necroptosis in ALS mice, we compared Ripk1, Ripk3, and Mlkl expression levels by qPCR in the lumbar spinal cord of Tg SOD1 G93A mice at two time points, ranging from early-symptomatic (i.e., 12 weeks of age) to near end-stage paralysis (i.e., 15 weeks of age; hereafter referred to as symptomatic), and in age-matched Tg SOD1 WT mice and NTg littermates. In early-symptomatic Tg SOD1 G93A mice, only Ripk3 mRNA was significantly upregulated as compared to agematched Tg SOD1 WT and NTg mice (Fig. 1A). In contrast, in symptomatic Tg SOD1 G93A mice, both Ripk1 and Ripk3 mRNA were significantly increased (Fig. 1B), whereas Mlkl mRNA levels remained unchanged.
To assess protein levels of these necroptosis factors, we first confirmed the specificity of two anti-RIPK1 antibodies in PMN cultures from WT E12.5 mouse embryos by silencing Ripk1 (78% of control as determined by RT-qPCR). With the anti-RIPK1 antibody AB_394014, the band detected on the Western blot at the expected RIPK1 molecular mass of 74 kDa was reduced (Fig. 1C). Similar results were obtained for the second anti-RIPK1 antibody AB_397831 (data not shown). Using these validated antibodies, we then ran Western blots of mouse spinal cord samples and found a significant increase in RIPK1 protein levels in symptomatic, but not early-symptomatic (data not shown) Tg SOD1 G93A mice in comparison with Tg SOD1 WT and NTg mice, in tissue samples processed with RIPA (Fig. 1C,D) and urea (data not shown) extraction buffers.
For RIPK3 antibody validation, we used the spleen as a positive control given the high expression levels of RIPK3 in this organ in WT mice (Wang et al., 2016). Spleen tissue extracts from NTg;Ripk3 ϩ/ϩ mice revealed two prominent bands, at ϳ45kDa and at ϳ55 kDa (Fig. 1E). In spleen tissue extracts from NTg;Ripk3 Ϫ/Ϫ mice, the ϳ55-kDa band, corresponding to the known molecular mass of Figure 2. RIPK1 expression in brain cortex from ALS patients. Postmortem motor cortex (Brodmann's area 4) from sporadic ALS, SOD1 ALS, and non-ALS human brains was homogenized and processed for RIPK1 protein expression. Western blotting for RIPK1 protein. Two different antibodies against RIPK1 were used (A, AB_397831; B, AB_394014). C, Quantification of RIPK1 protein levels. ␤-ACTIN, loading control. No significant differences were detected for RIPK1 between sporadic ALS, SOD1 ALS, and non-ALS human brain samples. Results are presented in a scatter dot plot. Line ϭ mean. Statistical analysis was performed via Student's t test: t (10) ϭ 0.579, p ϭ 0.575; n ϭ 4; non-ALS, n ϭ 6; sporadic ALS, and n ϭ 2; SOD1 ALS. RIPK3, was significantly reduced while the ϳ45-kDa band (probably possibly a cleavage product) disappeared (Fig.  1E). We did not observe a similar ϳ45-kDa band and detected only a faint and inconsistent ϳ55-kDa band in spinal cord lysates from Tg SOD1 G93A and NTg mice with RIPA buffer only (Fig. 1E). Of note, non-specific bands at ϳ47 kDa (with RIPA buffer; Fig. 1E) and ϳ52 kDa (with urea buffer; Fig. 1F) were prominent and consistently observed in both NTg and Tg SOD1 G93A mice.
To validate the anti-MLKL antibody, we used extracts from mouse NIH 3T3 cells, which express high levels of MLKL protein and observed a 54-kDa band corresponding to molecular mass of the MLKL (Fig. 1G,H). However, using this same antibody we did not detect this band in spinal cord of NTg or Tg SOD1 G93A mice with either RIPA or urea buffer (Fig. 1G,H). With urea extraction, we detected a band at ϳ52 kDa, which was below the validated ϳ54-kDa band in the NIH 3T3 lysate (Fig. 1H). To generate a positive control for p-MLKL, we induced necroptosis with TNF␣/ zVAD/SMAC in L929 cells as done by Hitomi et al. (2008). While the extracts from the necroptosis-induced L929 cells showed the expected ϳ54-kDa band using our anti-p-MLKL antibody, we did not detect any such band in spinal cords of NTg or Tg SOD1 G93A mice (Fig. 1I). These results led us to conclude that levels of RIPK3, MLKL and p-MLKL in spinal cord of NTg or Tg SOD1 G93A mice are below the detection limits of the validated antibodies in Western blotting. Thus, RIPK1 was the only necroptosis pathway protein unambiguously detected and upregulated in Tg SOD1 G93A mice. The discrepancy between levels of Ripk3 transcripts and protein needs further elucidation.

RIPK1 expression in human ALS brain samples
We next asked whether a similar RIPK1 upregulation is observed in postmortem tissue from ALS patients. For Figure 3. Ripk3 Ϫ/Ϫ MNs are resistant to Tg SOD1 G93A astrocyte-mediated toxicity. MNs, isolated from E12.5 Ripk3 ϩ/ϩ or Ripk3 Ϫ/Ϫ mice, were co-cultured on primary astrocyte monolayers from Tg SOD1 G93A or NTg mice for 7 d. A, Representative images of MNs assessed using SMI32 immunolabeling. Scale bar: 50 m. B, Quantification of MN number. Ripk3 ϩ/ϩ MN number was significantly reduced on SOD1 G93A astrocytes (p ϭ 0.0013) compared to NTg. Ripk3 Ϫ/Ϫ MN number did not differ between NTg or SOD1 G93A astrocytes and was significantly increased (p ϭ 0.0370) compared to Ripk3 ϩ/ϩ MN number on SOD1 G93A astrocytes. Results are presented as a mean Ϯ SEM. Statistical analysis was performed via two-way ANOVA; n ϭ 3 biological replicates per genotype. ‫ء‬p Յ 0.05; ‫‪p‬ءء‬ Յ 0.01. this purpose, we used brain homogenates from Brodmann's area 4, the primary motor cortex, from patients with sporadic ALS (n ϭ 6), SOD1 ALS (n ϭ 2), and agematched control patients (n ϭ 4). Patient information can be found in Table 1.
Using two different validated antibodies against RIPK1, we detected the expected ϳ74-kDa band. However, both antibodies showed that RIPK1 expression was faint, highly variable across all samples (Fig. 2) and not significantly different between ALS and controls. In addition, we did not observe any significant correlations (linear regression, R Յ 0.05, p Ն 0.75) between age at onset, duration of the disease, postmortem delay and RIPK1 expression levels on Western blotting (data not shown).

Ripk3 mediates MN death in an in vitro model of ALS
Previous studies have shown that RIPK1 contributes to MN death in in vitro models of ALS (Re et al., 2014). To expand characterization of the machinery of necroptosis in in vitro models, we determined the involvement of RIPK3 using constitutive knock-out mice (Ripk3 -/-) deficient in this kinase (Newton et al., 2004), which develop normally to adulthood without any observed defects in weight gain or fertility, and no histologic defects in major organs, including the CNS (Newton et al., 2004). We first assessed mRNA expression of Ripk3 in the brain and spinal cord from Ripk3 Ϫ/Ϫ mice and their WT counterparts. As expected, no expression of Ripk3 mRNA transcript was detected in Ripk3 Ϫ/Ϫ CNS (data not shown). To assess the role of Ripk3 in astrocyte-mediated MN toxicity in the SOD1 G93A model, we co-cultured MNs obtained from Ripk3 Ϫ/Ϫ and Ripk3 ϩ/ϩ mice with astrocytes from Tg SOD1 G93A and NTg mice (Nagai et al., 2007;Re et al., 2014;Ikiz et al., 2015) and monitored the loss of MN over time. We found that MNs deficient in Ripk3 survived significantly longer than WT MNs co-cultured with Tg SOD1 G93A astrocytes (Fig. 3). These results indicate that the constitutive deletion of Ripk3 protects embryonic MNs against the deleterious effects of SOD1 G93A astrocytes, similar to what has been shown for RIPK1 (Re et al., 2014). Therefore, both RIPK1 and RIPK3 appear to be significant contributors for MN death in this in vitro model of ALS. Of note, here, we did not test the potential effect of Ripk3 deletion in SOD1 G93A astrocytes on MN death, since our previous work (Re et al., 2014) established that RIPK1 contributes to motor neurodegeneration via a cell autonomous mechanism.

Genetic deletion of Ripk3 does not alter the pathologic hallmarks of Tg SOD1 G93A mice
Guided by these in vitro data, we expanded our investigation of necroptosis in ALS using an in vivo model of the disease. We posited that if the necroptosis contributes to neurodegeneration in Tg SOD1 G93A mice, then inhibition of this pathway should mitigate the ALS-like phenotype in these animals. To achieve this, we generated Ripk3 homozygous knock-out mice in the Tg SOD1 G93A background. We chose to use the Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ mouse as a model for several reasons: first, Ripk3 Ϫ/Ϫ mice have been shown to be resistant to a variety of necroptosis stimuli (Vanlangenakker et al., 2012;Geserick et al., 2015;Zhao et al., 2017); second, mutant Ripk3 Ϫ/Ϫ mice are healthy, while mutant Ripk1 Ϫ/Ϫ mice die soon after birth; and lastly, RIPK3 is increasingly recognized as more necroptosis-specific than RIPK1, which also triggers inflammatory responses through activation of the NFkB pathway (Festjens et al., 2007;Ofengeim and Yuan, 2013).

Negative Results
Ripk3 ϩ/ϩ mice seem to diverge, demonstrating statistical significance of the differences between these genotypes by increasing the number of mice per group would be of dubious value.
Next, we assessed the morphological hallmarks of the disease in Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ mice, limiting our analysis to the male mutants in which an improvement in onset and survival was observed. Typically, Tg SOD1 G93A mice exhibit ϳ50% MN loss in the lumbar spinal cord by end-stage (Higgins et al., 2002), preceded by extensive NMJ denervation (Fischer et al., 2004), especially in the TA muscle (Pun et al., 2006). The number of MNs in lumbar segments 4 and 5 of the mouse spinal cord, including those that innervate the TA muscle, was quantified at P140 (Fig. 6A), a timepoint where sig-nificant MN loss can be observed in the B6.Cg-Tg(SOD1‫ء‬G93A)1Gur/J mice. A two-way ANOVA showed no significant difference in the number of MNs at P140 Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ compared to Tg SOD1 G93A ;Ripk3 ϩ/ϩ male mice, although both groups had significantly fewer MNs than their NTg counterparts (Fig. 6B). In addition, no difference in MN number was found at P140 between NTg;Ripk3 Ϫ/Ϫ and NTg;Ripk3 ϩ/ϩ male mice (Fig. 6B). For the status of NMJ innervation in the TA muscles of Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ and Tg SOD1 G93A ;Ripk3 ϩ/ϩ male mice, an earlier time point (P120) was chosen, since NMJ denervation precedes MN cell death in Tg SOD1 G93A mice. Similar to MN counts, no difference between the number of innervated, denervated and partiallyinnervated NMJs (see Methods for definitions) was found . Statistical significant difference was detected between NTg (Ripk3 ϩ/ϩ and Ripk3 Ϫ/Ϫ ) and Tg (SOD1 G93A ;Ripk3 ϩ/ϩ and SOD1 G93A ;Ripk3 Ϫ/Ϫ ), p Ͻ 0.0001. No difference was detected between Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ and Tg SOD1 G93A ;Ripk3 ϩ/ϩ mice. Results are presented as a mean Ϯ SEM. Statistical analysis was performed via two-way ANOVA followed by Newman-Keuls post hoc test; n ϭ 3 biological replicates per genotype. C, Representative images of NMJ assessed by the expression of BTX (red, postsynaptic) and VAChT (green, presynaptic) in the TA muscle of P120 mice. Left column, Tg SOD1 G93A ;Ripk3 ϩ/ϩ . Right column, Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ mice. Scale bar: 100 m. D, Quantification of NMJs from 20-m sections (n ϭ ϳ100 NMJs). NMJs were categorized as innervated (complete colocalization of BTX and VAChT), partial (partial colocalization of BTX and VAChT), or denervated (no colocalization between BTX and VAChT) and are presented as a percentage of the total NMJ number counted. No difference was observed in the number of innervated, partially innervated, and denervated NMJs between Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ and Tg SOD1 G93A ;Ripk3 ϩ/ϩ mice. Results are presented as a mean Ϯ SEM. Statistical analysis was performed via unpaired Student's t test, two-tailed; n ϭ 3 biological replicates per genotype. ‫‪p‬ءءء‬ Յ 0.001. when comparing Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ to Tg SOD1 G93A ; Ripk3 ϩ/ϩ male mice (Fig. 6C,D). Therefore, the observed delay in onset and extension in survival of male Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ compared to male Tg SOD1 G93A ; Ripk3 ϩ/ϩ mice was not associated with improvements in either motor performance or neuropathological hallmarks of the disease.
Since no differences were detected in MN survival and muscle innervation between the Tg SOD1 G93A ;Ripk3 ϩ/ϩ and Tg SOD1 G93A ;Ripk3 Ϫ/Ϫ mice, we compared the motor axons myelin compaction. This idea was prompted by the report of Ito et al. (2016) that myelin decompaction may serve as a proxy of necroptosis in ALS. Therefore, as a first step, we used electron microscopy to examine lumbar spinal cord and ventral roots from NTg and earlysymptomatic Tg SOD1 G93A mice (Fig. 7). As expected and shown by others (Guo et al., 2010;Vinsant et al., 2013), early-symptomatic Tg SOD1 G93A mice showed ultrastructural changes in the ventral horn, funiculus, and roots including both shrunken and swollen MNs, large vacuoles and degenerating axons (Fig. 7B). In the case of spared myelinated fibers, the myelin sheath was preserved in the anterior funiculus and ventral roots in Tg SOD1 G93A mice (Fig. 7C,D). However, myelin preservation varied within as well as between mice, and myelin alterations reminiscent of myelin decompaction were observed in both Tg SOD1 G93A and NTg mice (Fig. 7E,F).
In addition, since the link between necroptosis and myelin decompaction was reported to be more pronounced in mutant Optn Ϫ/Ϫ than in Tg SOD1 G93A mice (Ito et al., 2016), we generated a mouse with homozygous germline deletion of the Optn gene (Fig. 8A). After verifying the complete knock-out of the optineurin protein in Optn Ϫ/Ϫ mice (Fig. 8B), we then monitored Optn Ϫ/Ϫ mice development and found no significant difference compared to their WT littermates in terms of newborn birth weight, appearance and behavior (data not shown). Furthermore, up to two years of age, we recorded no difference in survival and behavior between Optn Ϫ/Ϫ and Optn ϩ/ϩ mice (data not shown). Similar to Ito et al. (2016), we observed no difference in spinal MN number between one-year old Optn Ϫ/Ϫ mice and their aged-matched WT littermates (Fig. 8B,C). In contrast to Ito et al. (2016), we did not observe any denervation of the TA muscle and observed similar density of NMJs in one-year old Optn Ϫ/Ϫ mice as compared to their aged-matched WT littermates (Fig. 8D,E). We were unable to detect any genotypespecific alterations in myelin compaction in the ventral white matter (Fig. 8G) or in the ventral roots (Fig. 8H). Therefore, while OPTN loss of function has been a proposed mechanism by which mutations in OPTN may lead to ALS (Maruyama et al., 2010), our data argue that the constitutive loss of OPTN at the germline level is not sufficient for the generation of an ALS-like phenotype in mice.

Discussion
Necroptosis has been implicated in the pathogenesis of ALS. Here, to confirm the role of this form of PCD in MN disease, we assessed the expression of its three known core factors in two distinct animal models of ALS, and in ALS patient tissue. Transcripts for Ripk1, Ripk3, and Mlkl were all detected in mouse spinal cord of NTg, Tg SOD1 WT and Tg SOD1 G93A mice. However, only mRNA for Results are presented as a mean Ϯ SEM. Statistical analysis was performed via unpaired Student's t test; n ϭ 3 biological replicates per genotype. E, Representative images of NMJ, of the TA muscle, assessed by the expression of BTX (red, postsynaptic) and Syn (green, presynaptic) from one-year-old Optn Ϫ/Ϫ mice. Colocalization of BTX and Syn represents an innervated NMJ. Scale bar: 50 m. F, Quantification of innervated NMJs (%) in the Optn ϩ/ϩ , Optn ϩ/Ϫ , and Optn Ϫ/Ϫ mice. Results are presented as mean Ϯ SEM. Statistical bius et al., 2016), we believe that the reported myelin sheath decompaction is the result of technical variability rather than a reflection of the disease process.
Collectively, our in vivo data call for further investigations targeting key determinants of necroptosis in models of ALS. To minimize the risk of compensatory mechanisms, the elimination of these factors should be done conditionally, after the postnatal period, and not constitutively as done here and in Ito et al. (2016). It would also be critical to obtain sensitive and specific immunoreagents to detect the active form of the drivers of necroptosis, especially at a cellular level. Lastly, stable and brain permeant small molecules can now be used to inhibit RIPK3 and other determinants of necroptosis pharmacologically. It would thus be quite important to test such compounds in animal models of ALS to determine whether necroptosis plays a pathogenic role in ALS. This is what Ito et al. (2016) have done with the kinase inhibitor of RIPK1, Nec1s, but since that study only reports on onset of motor dysfunction and on myelin decompaction, we believe that the potential therapeutic role of targetting key determinants of necroptosis in ALS and related neurodegenerative disorders remains to be established.