Necroptosis activation in Alzheimer's disease

Journal name:
Nature Neuroscience
Volume:
20,
Pages:
1236–1246
Year published:
DOI:
doi:10.1038/nn.4608
Received
Accepted
Published online

Abstract

Alzheimer's disease (AD) is characterized by severe neuronal loss; however, the mechanisms by which neurons die remain elusive. Necroptosis, a programmed form of necrosis, is executed by the mixed lineage kinase domain-like (MLKL) protein, which is triggered by receptor-interactive protein kinases (RIPK) 1 and 3. We found that necroptosis was activated in postmortem human AD brains, positively correlated with Braak stage, and inversely correlated with brain weight and cognitive scores. In addition, we found that the set of genes regulated by RIPK1 overlapped significantly with multiple independent AD transcriptomic signatures, indicating that RIPK1 activity could explain a substantial portion of transcriptomic changes in AD. Furthermore, we observed that lowering necroptosis activation reduced cell loss in a mouse model of AD. We anticipate that our findings will spur a new area of research in the AD field focused on developing new therapeutic strategies aimed at blocking its activation.

At a glance

Figures

  1. Increase in necroptosis markers in AD human brains.
    Figure 1: Increase in necroptosis markers in AD human brains.

    (a) Representative western blots of proteins extracted from AD (n = 12 cases) and CTL (n = 11 cases) brains probed with the indicated antibodies. Full blots are shown in Supplementary Figure 2. (b,c) Quantitative analyses of the blots revealed elevated levels of RIPK1 (t(21) = 3.444, P = 0.002 for RIPA; t(21) = 2.205, P = 0.038 for urea) and MLKL (t(21) = 2.443, P = 0.023 for RIPA; t(21) = 3.126; P = 0.0047 for urea) in AD patients compared with CTL patients in both fractions. (d) Representative microphotographs of brain sections from AD (n = 14 cases) and CTL (n = 12 cases) patients immunostained with the indicated antibodies. (eg) Quantitative analyses of the immunoreactivity showed a significant increase of RIPK1 and MLKL in AD patients compared with CTL patients (t(24) = 3.447, P = 0.002 and t(24) = 2.667, P = 0.013, respectively). No significant difference was detected in RIPK3 levels (t(24 0.0001, P = 0.999). Quantification of western blots was determined by normalizing the protein of interest to β-actin. Data are presented as box plots, and were analyzed by unpaired t test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. *P < 0.05.

  2. Necrosome formation in AD.
    Figure 2: Necrosome formation in AD.

    (af) Microphotographs and quantitative analyses of brain sections from AD and CTL patients immunostained with the indicated antibodies. Quantitative analysis revealed that the number of colocalized pixels was significantly higher in AD patients than in CTL patients (b: t(28) = 3.270, P = 0.003; d: t(28) = 2.659, P = 0.013; f: t(28) = 2.437, P = 0.022). (g) Proteins extracted from AD and CTL brains were immunoprecipitated with an antibody against RIPK1 and probed for MLKL. The IgG band is shown as a control. Full blots are shown in Supplementary Figure 2. The graph shows the quantification of the western blots and highlights a stronger interaction between RIPK1 and MLKL in AD brains compared with CTL brains (t(12) = 2.542, P = 0.026). (h) Microphotographs of brain sections immunostained with a pMLKL specific antibody. (i) Quantitative analysis of the pMLKL immunohistochemistry showed a significant increase of pMLKL in AD patients compared with CTL patients (t(22) = 3.277 (P = 0.003). (j) Representative western blot probed with a MLKL antibody. The two bands represent MLKL monomers and dimers. Full blots are shown in Supplementary Figure 2. (k) Quantitative analyses of the MLKL blots were obtained by normalizing the intensity value of the dimers over the intensity value of the monomers (t(22) = 2.322, P = 0.030). (l) Microphotographs of brain sections from AD and CTL patients colabeled with the indicated antibodies. Statistical evaluation by Mander's correlation, followed by Costes randomization test revealed that, in CTL cases, 36.47 ± 1.4% of pMLKL immunoreactivity was in the membrane (R = 0.3288 and Costes P = 0.96). In AD cases, 42.31 ± 1.5% of pMLKL immunoreactivity was in the membrane (R = 0.360 and Costes P = 0.98). (m) Quantitative analyses of the pMLKL/cadherin colocalization showed that the number of colocalized pixels was significantly higher in AD cases compared with CTL cases (t(28) = 4.504, P = 0.0001). Data in b, d, f, g, i, k and m are presented as box plots and were analyzed by unpaired t test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. n = 15 cases for CTL and n = 15 cases for AD for af,l,m; n = 11 cases for CTL and n = 13 cases for AD for hk; n = 7 cases for CTL and n = 7 cases for AD for g. *P < 0.05.

  3. Necroptosis activation is linked to reduced brain weight.
    Figure 3: Necroptosis activation is linked to reduced brain weight.

    (ac) Boxplot of log2 expression values in AD and CTL patients for genes RIPK1, RIPK3 and MLKL, respectively. The data indicate that RIPK1 and MLKL levels were significantly higher in AD compared with CTL cases (t(193) = 6.890, P = 7.5 × 10−11; t(193) = 7.017, P = 3.7 × 10−11, respectively). In contrast, RIPK3 levels were not different between the two groups (t(193) = −0.247, P = 0.805). (d) Scatterplot and regression line for the regression (without covariates) between RIPK1 and brain weight with 95% confidence intervals. Data in ac are presented as box plots and were analyzed by moderated t test (n = 98 cases for CTL and n = 97 cases for AD). Data in d were analyzed by linear regression (n = 93 cases for AD). In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. *P < 0.05.

  4. The activation of RIPK1 and MLKL correlates with Braak stage.
    Figure 4: The activation of RIPK1 and MLKL correlates with Braak stage.

    (ac) Boxplot of log2 expression values in AD patients for each Braak stage for RIPK1, RIPK3 and MLKL, respectively. RIPK1 and MLKL expression levels positively correlate with Braak stage (t(92) = 2.107, P = 0.035; t(92) = 2.488, P = 0.013, respectively). RIPK3 levels did not correlate with Braak stage (t(92) = 0.677, P = 0.499). Data were analyzed by ordinal logistical regression and presented as box plots. Data were analyzed by ordinal logistical regression and are presented as box plots. In the box plots, the center line represents the median value, the limits represent the 25th (Q1) and 75th (Q3) percentile, the whiskers represent the minimum and maximum value of the distribution, and the points represents the individual experimental value. The points outside the whiskers were defined by the interquartile range (IQR) rule, meaning they fall below Q1 – 1.5 IQR or above the Q3 + 1.5 IQR. (d,e) Microphotographs of AD brain sections colabeled with the indicated antibodies. Statistical evaluation by Mander's correlation followed by Costes randomization test indicated that 55.48 ± 2.8% and 46.85 ± 4.0% of the AT8 immunoreactivity colocalized with RIPK1 (R = 0.605, Costes P = 0.99) and pMLKL (R = 0.529, Costes P = 0.98), respectively. (fi) Results for the quantile regression between MMSE and the expression levels of the three necroptotic markers, RIPK1, RIPK3 and MLKL. The scatter plots show the regression coefficients as a function of percentiles and the standard errors. For RIPK1, after FDR correction there was a significant negative regression for percentiles ranging from 30th to the 40th percentiles. For RIPK3, no significant correlation was detected for any of the percentiles. For MLKL, after FDR correction there was a significant negative regression for all percentiles ranging from the 30th to the 50th percentiles. (g) Results for the quantile regression between MMSE and RIPK1:MLKL. After FDR correction, the interaction RIPK1:MLKL negatively correlated with MMSE for a wider range of percentiles (20th-50th). n = 94 AD cases for ac, n = 15 for d,e and n = 62 for fi.

  5. Regulation of AD transcriptome and risk-associated genes by RIPK1.
    Figure 5: Regulation of AD transcriptome and risk-associated genes by RIPK1.

    Genes regulated by RIPK1 overlapped significantly with multiple AD expression profiles and brain disease risk loci. Interactions detected in either anterior prefrontal cortex or ectorhinal cortex are shown. Red edges indicate genes positively regulated by RIPK1, blue denotes negative regulation. ADHD, attention deficit hyperactivity disorder; ALS, amyotrophic lateral sclerosis; ASD, autism spectrum disorder; ID, intellectual disability; SCZ, schizophrenia.

  6. Necroptosis activation exacerbates cognitive deficits in APP/PS1 mice.
    Figure 6: Necroptosis activation exacerbates cognitive deficits in APP/PS1 mice.

    (a,b) Learning curves of mice trained in Morris water maze (NonTg-GFP, n = 14 mice; NonTg-MLKL, n = 12 mice; APP/PS1-GFP, n = 14 mice; APP/PS1-MLKL, n = 14 mice). For the escape latency, day effect (F(4, 250) = 31.26; P < 0.0001), group effect (F(3, 250) = 21.37; P < 0.001) and group × day interaction (F(12, 250) = 2.16; P = 0.014). For distance traveled, day effect (F(4, 250) = 45.48; P < 0.0001), group effect (F(34, 250) = 31.46; P < 0.001) and group × day interaction (F(12, 250) = 1.87; P = 0.040). Post hoc tests indicated that the escape latency for APP/PS1-MLKL mice was significantly different than that for NonTg-GFP mice at days 3, 4 and 5 (shown by #); it was significantly different than that for NonTg-MLKL and APP/PS1-GFP mice at day 5 (shown by ## and ###, respectively). Post hoc tests indicated that the distance traveled for APP/PS1-MLKL mice was significantly different than NonTg-GFP mice at days 2–5 (shown by #); it was significantly different than NonTg-MLKL mice at days 3 and 4 (shown by ##); it was significantly higher than APP/PS1-GFP mice at days 2, 4 and 5 (shown by ###). (c) Number of platform location crosses during a single 60-s probe trial (F(3, 50) = 11.59; P < 0.0001). Post hoc tests showed that NonTg-GFP performed significantly better than the other three groups (shown by ). In addition, APP/PS1-MLKL performed significantly worse than the other three groups (shown by ). (d) Number of platform location crosses analyzed as a percentage change indicated that the slopes for the two groups were significantly different from each other (F(1, 38) = 4.67; P = 0.037). (e) Swim speed measured during a single 60-s probe trial. The values were not statistically significant among the groups (F(3, 50) = 1.164; P = 0.333). (f,g) Microphotographs of NonTg brain sections injected with the GFP or the MLKL virus, respectively. Sections were stained with an anti-GFP antibody to visualize viral diffusion. (hj) Sections from APP/PS1-GFP mice (n = 8) were stained with the indicated antibodies. Mander's correlation analysis indicated a 66.84 ± 3.2% colocalization between GFP and the neuronal marker NeuN, 20.06 ± 3.0% colocalization between GFP and the astrocytic marker GFAP and 31.14 ± 2.4% colocalized between GFP and the microglial marker Iba1. (k) Quantitative analysis of the colocalized pixels indicated that most of the virus-infected neurons. One-way ANOVA indicated that these values were significantly different among each other (F(2, 23) = 70.35; P < 0.001). Bonferroni's multiple comparison test indicated that all three groups were statistically different from each other. Data in a,b were analyzed by two-way ANOVA and are presented by mean ± s.e.m.; data in c,e,k were analyzed by one-way ANOVA and are presented as box plots. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. Bonferroni's was used for post hoc tests. Data in d were analyzed by linear regression and are presented as mean ± s.e.m.

  7. Constitutively active MLKL induces a higher degree of neuronal death in APP/PS1 than NonTg mice.
    Figure 7: Constitutively active MLKL induces a higher degree of neuronal death in APP/PS1 than NonTg mice.

    (a) Representative CA1 sections from APP/PS1 and NonTg mice injected with the MLKL or GFP AAVs and stained with NeuN, a neuronal marker. (b) Quantitative analyses of the stained sections (n = 18 pictures per group; 3 pictures per mouse, 6 mice per group). Data were analyzed by one-way ANOVA and presented as box plots. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. Analyses revealed a significant difference among the four groups (F(3, 67) = 21.77; P < 0.0001). Bonferroni's post hoc analyses indicated that all the pairwise comparisons were significantly different except for NonTg-GFP and APP/PS1-GFP. #P < 0.05 between the two NonTg groups; ##P < 0.05 between the two APP/PS1 groups. (c) Analysis of the stained sections presented as a percentage change. Data were analyzed by linear regression, which indicated that the slopes for the two groups were significantly different from each other (F(1, 67) = 4.79; P = 0.032). Data are presented as mean ± s.e.m.

  8. Necroptosis contributes to neuronal death in 5xFAD mice.
    Figure 8: Necroptosis contributes to neuronal death in 5xFAD mice.

    (a) Primary neurons from APP/PS1 and wild-type (WT) mice were treated with Nec1S or vehicle (APP/PS1, n = 36 wells from 9 mice per drug per time point; WT, n = 12 wells from 3 mice per drug per time point). The graph shows the number of NeuN-positive neurons after 11 and 15 DIV. There was a significant effect for time (F(1,184) = 76.13; P < 0.0001) and groups (F(3,184) = 7.109; P = 0.0002) as well as a significant time × group interaction (F(3,184) = 7.109; P = 0.0002). (b) The graph shows the quantitative analyses of pMLKL/MLKL measured by western blot (APP/PS1-vehicle, n = 5; APP/PS1-Nec1S, n = 5; WT-vehicle, n = 4; WT-Nec1S, n = 4). Analyses revealed a significant difference among the four groups (F(3,14) = 10.20; P = 0.0008). Bonferroni's post hoc analyses revealed that all of the APP/PS1-vehicle group was significantly different from the other three groups. (c) Primary neurons from APP/PS1 and wild-type mice were infected with an AAV-expressing GFP and then treated with Nec1S or vehicle (APP/PS1, n = 72 wells from 9 mice per drug per time point; WT, n = 16 wells from 2 mice per drug per time point). The graph shows the GFP fluorescence after 11 and 15 DIV. There was a significant effect for time (F(1,344) = 170.2; P < 0.0001) and groups (F(3,344) = 102.4; P < 0.0001) as well as a significant time × group interaction (F(3,344) = 78.43; P < 0.0001). (d) The graph shows the quantitative analyses of pMLKL/MLKL, measured by western blot (APP/PS1-vehicle, n = 5; APP/PS1-Nec1S, n = 5; WT-vehicle, n = 4; WT-Nec1S, n = 4). Analyses revealed a significant difference among the four groups (F(3,14) = 11.30; P = 0.0005). Bonferroni's post hoc analyses indicated that all the groups were significantly different from the APP/PS1-vehicle. (e) Representative brain sections from 5xFAD mice treated with Nec1S or vehicle stained with Fluoro-Jade. (f) Quantitative analyses of the Fluoro-Jade staining revealed a significant difference between groups (t(7) = 4.353; P = 0.0033). (g) The graph shows the quantitative analyses of pMLKL/MLKL measured by western blot (5xFAD-vehicle, n = 4, 5xFAD-Nec1s, n = 4). Analyses revealed a significant difference between groups (t(6) = 2.627; P = 0.0341). Data in a and c are presented as mean ± s.e.m. and were analyzed by two-way ANOVA. Data in b and d are presented as box plots and were analyzed by one-way ANOVA. Data in f,g are presented as box plots and were analyzed by unpaired t test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. *P < 0.05, **P < 0.01, ***P < 0.001.

  9. Levels of necroptotic markers in TBS and Triton fractions
    Supplementary Fig. 1: Levels of necroptotic markers in TBS and Triton fractions

    (a) Representative western blots of TBS and Triton extracts from AD and CTL patients probed with the indicated antibodies. (b-d) Quantitative analyses of the western blots. RIPK1 was not detected in the TBS fraction. For all the proteins measured, no changes were detected between the two groups. For RIPK1 triton [t(21) = 0.840, P = 0.409]; for RIPK3 TBS [t(21) = 0.402, P = 0.691]; for RIPK3 triton [t(21) = 0.357, P = 0.724]; for MLKL TBS [t(21) = 0.176, P = 0.862], for MLKL triton [t(21) = 0.065, P = 0.9491]. Data were normalized to β-actin, used as a loading control. Data are presented as box plots and were analyzed by unpaired t-test. n = 11 CTL cases and n = 12 AD cases. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  10. Full blots of RIPK1 and MLKL western blots in human cases
    Supplementary Fig. 2: Full blots of RIPK1 and MLKL western blots in human cases

    (a-b) Proteins extracted with RIPA and UREA buffer from CTL and AD cases. Blots were probed with the indicated antibodies. The levels of the protein of interest (RIPK1 or MLKL) were normalized to β-actin for every sample, then all samples were expressed as a ratio with respect to the average of the CTL samples in the same blot. Doing so, the AD samples are expressed as a percentage change over the CTL samples. (c) Proteins from CTL and AD cases were immunoprecipitated with a RIPK1 antibody and probed with an MLKL antibody. The black arrows point to the MLKL band, the black arrowheads point to the IgG. (d) Proteins from CTL and AD cases were run in not reducing conditions and probed with an MLKL antibody. The gray arrows point to the MLKL dimers, the gray arrowheads point to the MLKL monomers. The levels of MLKL dimers were normalized to the levels of MLKL monomers for every samples. Then all the samples in the same blot were expressed as ratio with respect to the average of the CTL sample in the same blot. Doing so, the AD samples are expressed as percentage change over the CTL samples.

  11. Increased pMLKL in AD colocalize with phosphorylated tau
    Supplementary Fig. 3: Increased pMLKL in AD colocalize with phosphorylated tau

    (a) Representative confocal images from CTL and AD cases stained with a different pMLKL antibody than the one used for Fig. 2. (b) The graph shows the quantitative analyses of the pMLKL immunoreactivity tissue [t(28) = 4.561; P < 0.0001]. (c) Representative confocal images from CTL and AD cases stained with the indicated antibodies. Statistical evaluation by Mander’s correlation, followed by Costes randomization test indicates that in CTL cases, 36.04 ± 1.5% of pMLKL immunoreactivity was located in the membrane [R(13) = 0.319 and Costes P = 0.96]. In AD cases, 52.90 ± 3.2% of pMLKL immunoreactivity was located in the membrane [R(13) = 0.365 and Costes P = 0.97]. (d) The graph shows the quantitative analyses of the colocalized pMLKL and cadherin pixels [t(28) = 6.991; P < 0.0001]. (e) Sections from AD patients were stained with the indicated antibodies. Statistical evaluation by Mander’s correlation followed by Costes randomization test indicates that 39.17 ± 3.1% of pMLKL immunoreactivity co-localized with CP13 [R(13) = 0.4155 and Costes p = 0.99]. These data confirm the data shown in Fig. 4, using a different pMLKL antibody. For all the data shown here, n = 15 CTL cases and n = 15 AD cases. Data in panels b and d were analyzed by unpaired t-test and are presented as box plots. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  12. Caspase-3/pMLKL colocalization is similar between CTL and AD cases
    Supplementary Fig. 4: Caspase-3/pMLKL colocalization is similar between CTL and AD cases

    (a) Microphotographs of CTL (n = 15 cases) and AD (n = 15 cases) brains stained with the indicated antibodies. (b), Quantitative analysis of the sections, which was obtained by Mander’s correlation followed by Costes randomization test indicates that 42.10 ± 1.9% of pMLKL immunoreactivity co-localized with Caspase-3 [R(13) = 0.6159 and Costes P = 0.965] for CTL, and 45.53 ± 2.3% [R(13) = 0.6075 and Costes P = 0.970] for AD. There was no significant difference between the two groups [t(28) = 0.319; P = 0.752). Data are presented as box plots and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  13. pMLKL is mainly found in neurons
    Supplementary Fig. 5: pMLKL is mainly found in neurons

    (a-c) Microphotographs of AD brain (n = 15) sections co-labeled with the indicated antibodies. Statistical evaluation by Mander’s correlation followed by Costes randomization test indicates that 60.22 ± 3.3% of pMLKL immunoreactivity co-localized with NeuN [R(13) = 0.583 and Costes P = 0.99], 11.14 ± 1.4% co-localized with GFAP [R(13) = 0.179 and Costes P = 0.63], and 28.00 ± 2.6% co-localized with Iba1 [R(13) = 0.578 and Costes P = 0.96].

  14. Schematic representation of the experimental design used to perform the causal inference testing.
    Supplementary Fig. 6: Schematic representation of the experimental design used to perform the causal inference testing.

    Gene regulatory network for RIPK1 based inferred from postmortem brain tissue samples. Causal inference testing was used to determine directed regulatory links between RIPK1 and its correlated genes.

  15. Full blots showing levels of necroptotic markers in 5xFAD and APP/PS1 mice
    Supplementary Fig. 7: Full blots showing levels of necroptotic markers in 5xFAD and APP/PS1 mice

    (a) Proteins from 5xFAD mice (n = 5 mice) and littermate controls (NT; n = 7 mice), and APP/PS1 mice (n = 8) and littermate controls (WT; n = 8), were probed with the indicated antibodies. (b-d) Quantitative analyses of the blots show an increase of the kinases in the 5xFAD mice compared to control [t(10) = 2.423, P = 0.036 for RIPK1; t(10) = 2.910, P = 0.016 for MLKL; t(10) = 3.076, P = 0.012 for pMLKL]. No differences were found in the APP/PS1 mice compared to their control [t(14) = 1.245, P = 0.234 for RIPK1; t(14) = 0.807, P = 0.433 for MLKL]. (e-f) Representative confocal microphotographs of sections from 5xFAD and APP/PS1 stained with Fluro-Jade. Quantification of western blots were obtained by normalizing the protein of interest to β-actin (used as a loading control). Data are presented as box plots and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  16. Increasing necroptosis does not change A[beta] and tau pathology
    Supplementary Fig. 8: Increasing necroptosis does not change Aβ and tau pathology

    (a-b) Representative hippocampal sections from APP/PS1 mice injected with the AAV expressing GFP or MLKL. Sections were stained with an Aβ42-specific antibody (n = 14 mice per group). (c-f) The graphs show soluble and insoluble Aβ40 and Aβ42 levels measured by sandwich ELISA. Panel c, t(26) = 1.197, P = 0.242. Panel d, t(26) = 0.4513, P = 0.655. Panel e, t(26) = 0.6518, P = 0.520. Panel f, t(26) = 0.8277, P = 0.415. Data are presented as box plots, and were analyzed by unpaired t-test (n = 14 mice per group). (g-j) Western blots of proteins extracted from APP/PS1-GFP (n = 7 mice) and APP/PS1-MLKL mice (n = 8 mice). Blots were probed with the indicated antibodies. The levels of Tau-5, which recognize total mouse tau, were similar between the two groups [t(13) = 0.484; P = 0.637]. The CP13 antibody, which is raised against tau phosphorylated at Ser202, recognized two bands of ~60 and ~50 kDa. Statistical analyses of both bands indicated that CP13 levels were similar between the two groups [t(13) = 0.48; P = 0.64] for both bands. Data are presented as box plots, and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  17. Full blots for necrostatin treatments
    Supplementary Fig. 9: Full blots for necrostatin treatments

    (a-b) Proteins extracted from APP/PS1 and wild type primary neurons used for NeuN staining. Quantitative analyses are shown in Fig. 8b. (c-d) Proteins extracted from APP/PS1 and wild type primary neurons transfected with AAV-GFP. Quantitative analyses are shown in Fig. 8d. (e) Proteins extracted from 5xFAD and wild type mice. Quantitative analyses are shown in Fig. 8g. Blots were probed with the indicated antibodies.

  18. Validation of the RIPK1, RIPK3, MLKL, and pMLKL antibodies
    Supplementary Fig. 10: Validation of the RIPK1, RIPK3, MLKL, and pMLKL antibodies

    (a) Proteins extracted from wild type cells, RIPK1 knockout cells (as a negative control), RIPK1 knockout cells transfected with a RIPK1 expressing plasmid (as a positive control), CTL and AD human cases, wild type and 5xFAD mice were probed with the RIPK1 antibody. The expected band of 73 kDa (arrow) was not present in the knockout cells. (b) Proteins extracted from wild type mice, RIPK3 knockout mice (as a negative control), wild type cells transfected with a RIPK3 expressing plasmid (as a positive control), CTL and AD human cases, wild type and 5xFAD mice, were probed with the RIPK3 antibody. The expected band of 55 kDa (arrow) was not present in the RIPK3 knockout mice and was present in the cells transfected with the RIPK3-expressing plasmid. The RIPK3 band in the positive control ran a little slower as the plasmid had a GFP tag to its C-terminal. (c) To validate the MLKL antibody, we loaded on a gel proteins extracted from wild type cells, MLKL knockout cells (as a negative control), MLKL knockout cells transfected with a MLKL-expressing plasmid (as a positive control), CTL and AD human cases, non-transgenic and 5xFAD mice. The expected band of 51 kDa (arrow) was not present in the knockout cells, but it was present when these cells were transfected with a MLKL plasmid. (d) To validate the phospho-specific MLKL antibody, we loaded on a gel protein extracted from MLKL knockout and wild type cells. As a positive control, cells were treated with 1 ng/mL TNFα and 50 μM Caspase inhibitor Z-VAD-FMK to induce necroptosis. (e) To validate the RIPK1 and MLKL antibodies for immunohistochemistry, we used HAP1 cells where the respective genes were knocked out. As a control (WT) we used the parental cell line. To validate the RIPK3 antibody for immunohistochemistry, we used mouse primary fibroblasts isolated from RIPK3 knockout and wild type mice. (f) To validate the pMLKL antibody for immunohistochemistry, we used HAP-1 cells and induced necroptosis activation.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

NCBI Reference Sequence

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Author information

  1. These authors contributed equally to this work.

    • Antonella Caccamo &
    • Caterina Branca

Affiliations

  1. Arizona State University-Banner Neurodegenerative Disease Research Center at the Biodesign Institute, Arizona State University, Tempe, Arizona, USA.

    • Antonella Caccamo,
    • Caterina Branca,
    • Eric Ferreira,
    • Ramona Belfiore,
    • Wendy Winslow &
    • Salvatore Oddo
  2. Translational Genomics Research Institute, Phoenix, Arizona, USA.

    • Ignazio S Piras,
    • Matthew J Huentelman &
    • Winnie S Liang
  3. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Ben Readhead &
    • Joel T Dudley
  4. Department of Neurobiology and Behavior, University of California, Irvine, Irvine, California, USA.

    • Elizabeth E Spangenberg &
    • Kim N Green
  5. Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.

    • Ramona Belfiore
  6. School of Life Sciences, Arizona State University, Tempe, Arizona, USA.

    • Salvatore Oddo

Contributions

A.C. and C.B. designed and performed the experiments and analyzed the data. I.S.P. and M.J.H. performed the statistical analyses. E.F. performed the confocal imaging and quantification. W.S.L. generated the expression data from the microarray analyses used to generate the RIPK1 causal regulatory network. B.R. and J.T.D. generated the RIPK1 causal regulatory network and performed the associated gene set analysis. E.E.S. and K.N.G. performed the experiments on 5xFAD mice. R.B. performed the colocalization experiments described in Supplementary Figure 5. W.W. performed the co-immunoprecipitation experiments. S.O. conceptualized and designed the experiments, analyzed the data, and wrote the manuscript. All of the authors contributed to the preparation of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Levels of necroptotic markers in TBS and Triton fractions (183 KB)

    (a) Representative western blots of TBS and Triton extracts from AD and CTL patients probed with the indicated antibodies. (b-d) Quantitative analyses of the western blots. RIPK1 was not detected in the TBS fraction. For all the proteins measured, no changes were detected between the two groups. For RIPK1 triton [t(21) = 0.840, P = 0.409]; for RIPK3 TBS [t(21) = 0.402, P = 0.691]; for RIPK3 triton [t(21) = 0.357, P = 0.724]; for MLKL TBS [t(21) = 0.176, P = 0.862], for MLKL triton [t(21) = 0.065, P = 0.9491]. Data were normalized to β-actin, used as a loading control. Data are presented as box plots and were analyzed by unpaired t-test. n = 11 CTL cases and n = 12 AD cases. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  2. Supplementary Figure 2: Full blots of RIPK1 and MLKL western blots in human cases (348 KB)

    (a-b) Proteins extracted with RIPA and UREA buffer from CTL and AD cases. Blots were probed with the indicated antibodies. The levels of the protein of interest (RIPK1 or MLKL) were normalized to β-actin for every sample, then all samples were expressed as a ratio with respect to the average of the CTL samples in the same blot. Doing so, the AD samples are expressed as a percentage change over the CTL samples. (c) Proteins from CTL and AD cases were immunoprecipitated with a RIPK1 antibody and probed with an MLKL antibody. The black arrows point to the MLKL band, the black arrowheads point to the IgG. (d) Proteins from CTL and AD cases were run in not reducing conditions and probed with an MLKL antibody. The gray arrows point to the MLKL dimers, the gray arrowheads point to the MLKL monomers. The levels of MLKL dimers were normalized to the levels of MLKL monomers for every samples. Then all the samples in the same blot were expressed as ratio with respect to the average of the CTL sample in the same blot. Doing so, the AD samples are expressed as percentage change over the CTL samples.

  3. Supplementary Figure 3: Increased pMLKL in AD colocalize with phosphorylated tau (620 KB)

    (a) Representative confocal images from CTL and AD cases stained with a different pMLKL antibody than the one used for Fig. 2. (b) The graph shows the quantitative analyses of the pMLKL immunoreactivity tissue [t(28) = 4.561; P < 0.0001]. (c) Representative confocal images from CTL and AD cases stained with the indicated antibodies. Statistical evaluation by Mander’s correlation, followed by Costes randomization test indicates that in CTL cases, 36.04 ± 1.5% of pMLKL immunoreactivity was located in the membrane [R(13) = 0.319 and Costes P = 0.96]. In AD cases, 52.90 ± 3.2% of pMLKL immunoreactivity was located in the membrane [R(13) = 0.365 and Costes P = 0.97]. (d) The graph shows the quantitative analyses of the colocalized pMLKL and cadherin pixels [t(28) = 6.991; P < 0.0001]. (e) Sections from AD patients were stained with the indicated antibodies. Statistical evaluation by Mander’s correlation followed by Costes randomization test indicates that 39.17 ± 3.1% of pMLKL immunoreactivity co-localized with CP13 [R(13) = 0.4155 and Costes p = 0.99]. These data confirm the data shown in Fig. 4, using a different pMLKL antibody. For all the data shown here, n = 15 CTL cases and n = 15 AD cases. Data in panels b and d were analyzed by unpaired t-test and are presented as box plots. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  4. Supplementary Figure 4: Caspase-3/pMLKL colocalization is similar between CTL and AD cases (310 KB)

    (a) Microphotographs of CTL (n = 15 cases) and AD (n = 15 cases) brains stained with the indicated antibodies. (b), Quantitative analysis of the sections, which was obtained by Mander’s correlation followed by Costes randomization test indicates that 42.10 ± 1.9% of pMLKL immunoreactivity co-localized with Caspase-3 [R(13) = 0.6159 and Costes P = 0.965] for CTL, and 45.53 ± 2.3% [R(13) = 0.6075 and Costes P = 0.970] for AD. There was no significant difference between the two groups [t(28) = 0.319; P = 0.752). Data are presented as box plots and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  5. Supplementary Figure 5: pMLKL is mainly found in neurons (671 KB)

    (a-c) Microphotographs of AD brain (n = 15) sections co-labeled with the indicated antibodies. Statistical evaluation by Mander’s correlation followed by Costes randomization test indicates that 60.22 ± 3.3% of pMLKL immunoreactivity co-localized with NeuN [R(13) = 0.583 and Costes P = 0.99], 11.14 ± 1.4% co-localized with GFAP [R(13) = 0.179 and Costes P = 0.63], and 28.00 ± 2.6% co-localized with Iba1 [R(13) = 0.578 and Costes P = 0.96].

  6. Supplementary Figure 6: Schematic representation of the experimental design used to perform the causal inference testing. (353 KB)

    Gene regulatory network for RIPK1 based inferred from postmortem brain tissue samples. Causal inference testing was used to determine directed regulatory links between RIPK1 and its correlated genes.

  7. Supplementary Figure 7: Full blots showing levels of necroptotic markers in 5xFAD and APP/PS1 mice (294 KB)

    (a) Proteins from 5xFAD mice (n = 5 mice) and littermate controls (NT; n = 7 mice), and APP/PS1 mice (n = 8) and littermate controls (WT; n = 8), were probed with the indicated antibodies. (b-d) Quantitative analyses of the blots show an increase of the kinases in the 5xFAD mice compared to control [t(10) = 2.423, P = 0.036 for RIPK1; t(10) = 2.910, P = 0.016 for MLKL; t(10) = 3.076, P = 0.012 for pMLKL]. No differences were found in the APP/PS1 mice compared to their control [t(14) = 1.245, P = 0.234 for RIPK1; t(14) = 0.807, P = 0.433 for MLKL]. (e-f) Representative confocal microphotographs of sections from 5xFAD and APP/PS1 stained with Fluro-Jade. Quantification of western blots were obtained by normalizing the protein of interest to β-actin (used as a loading control). Data are presented as box plots and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  8. Supplementary Figure 8: Increasing necroptosis does not change Aβ and tau pathology (474 KB)

    (a-b) Representative hippocampal sections from APP/PS1 mice injected with the AAV expressing GFP or MLKL. Sections were stained with an Aβ42-specific antibody (n = 14 mice per group). (c-f) The graphs show soluble and insoluble Aβ40 and Aβ42 levels measured by sandwich ELISA. Panel c, t(26) = 1.197, P = 0.242. Panel d, t(26) = 0.4513, P = 0.655. Panel e, t(26) = 0.6518, P = 0.520. Panel f, t(26) = 0.8277, P = 0.415. Data are presented as box plots, and were analyzed by unpaired t-test (n = 14 mice per group). (g-j) Western blots of proteins extracted from APP/PS1-GFP (n = 7 mice) and APP/PS1-MLKL mice (n = 8 mice). Blots were probed with the indicated antibodies. The levels of Tau-5, which recognize total mouse tau, were similar between the two groups [t(13) = 0.484; P = 0.637]. The CP13 antibody, which is raised against tau phosphorylated at Ser202, recognized two bands of ~60 and ~50 kDa. Statistical analyses of both bands indicated that CP13 levels were similar between the two groups [t(13) = 0.48; P = 0.64] for both bands. Data are presented as box plots, and were analyzed by unpaired t-test. In the box plots, the center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution.

  9. Supplementary Figure 9: Full blots for necrostatin treatments (216 KB)

    (a-b) Proteins extracted from APP/PS1 and wild type primary neurons used for NeuN staining. Quantitative analyses are shown in Fig. 8b. (c-d) Proteins extracted from APP/PS1 and wild type primary neurons transfected with AAV-GFP. Quantitative analyses are shown in Fig. 8d. (e) Proteins extracted from 5xFAD and wild type mice. Quantitative analyses are shown in Fig. 8g. Blots were probed with the indicated antibodies.

  10. Supplementary Figure 10: Validation of the RIPK1, RIPK3, MLKL, and pMLKL antibodies (533 KB)

    (a) Proteins extracted from wild type cells, RIPK1 knockout cells (as a negative control), RIPK1 knockout cells transfected with a RIPK1 expressing plasmid (as a positive control), CTL and AD human cases, wild type and 5xFAD mice were probed with the RIPK1 antibody. The expected band of 73 kDa (arrow) was not present in the knockout cells. (b) Proteins extracted from wild type mice, RIPK3 knockout mice (as a negative control), wild type cells transfected with a RIPK3 expressing plasmid (as a positive control), CTL and AD human cases, wild type and 5xFAD mice, were probed with the RIPK3 antibody. The expected band of 55 kDa (arrow) was not present in the RIPK3 knockout mice and was present in the cells transfected with the RIPK3-expressing plasmid. The RIPK3 band in the positive control ran a little slower as the plasmid had a GFP tag to its C-terminal. (c) To validate the MLKL antibody, we loaded on a gel proteins extracted from wild type cells, MLKL knockout cells (as a negative control), MLKL knockout cells transfected with a MLKL-expressing plasmid (as a positive control), CTL and AD human cases, non-transgenic and 5xFAD mice. The expected band of 51 kDa (arrow) was not present in the knockout cells, but it was present when these cells were transfected with a MLKL plasmid. (d) To validate the phospho-specific MLKL antibody, we loaded on a gel protein extracted from MLKL knockout and wild type cells. As a positive control, cells were treated with 1 ng/mL TNFα and 50 μM Caspase inhibitor Z-VAD-FMK to induce necroptosis. (e) To validate the RIPK1 and MLKL antibodies for immunohistochemistry, we used HAP1 cells where the respective genes were knocked out. As a control (WT) we used the parental cell line. To validate the RIPK3 antibody for immunohistochemistry, we used mouse primary fibroblasts isolated from RIPK3 knockout and wild type mice. (f) To validate the pMLKL antibody for immunohistochemistry, we used HAP-1 cells and induced necroptosis activation.

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  1. Supplementary Text and Figures (2,389 KB)

    Supplementary Figures 1–10 and Supplementary Tables 1–5

  2. Supplementary Methods Checklist (525 KB)

Excel files

  1. Supplementary Data Set 1 (18 KB)

    Gene Expression File 1

  2. Supplementary Data Set 2 (322 KB)

    Gene Expression File 2

  3. Supplementary Data Set 3 (515 KB)

    Gene Expression File 3

Additional data