EMBO/SourceData huggingface dataset:
Figure 1B. Blots showing expression of hTauAT (hTau, Mr ~67 kDa) and/or endogenous mouse Tau (mTau, Mr ~45-55 kDa) in the hippocampus of 10 month old heterozygous (+/-) and homozygous (+/+) hTauAT mice in comparison to age matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as loading control.C. Quantification of (B). Ratio of hTau / mTau indicating a higher hTauAT -expression in homozygous mice (~1.8) compared to heterozygous mice (~1.0). Each bar represents an average of n=4 animals, error bars represent SEM.D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group).E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauATmice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity.F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3.G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau).H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal neuroncell bodies (arrows).I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.
Figure 2A. Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads).B. Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 month of age compared to non-reactive control littermate mice. In homozygous hTauAT mice the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates.C. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice.D. Quantification of (C). The ratio of hTauAT /mTau indicates a stronger aggregation in the homozygous hTauAT mice (~1.4) compared to heterozygous hTauAT mice (~0.7 (n=4 animals, error bars represent SEM).E. Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. (ds) dendriticspine, (pt) presynaptic terminal and (m) mitochondrium.F. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months.G. Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d).H. Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow).I. Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow).J. Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks).
Figure 3A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown.C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image).D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures.E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.
Figure 4A. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.For an overview of drug concentrations see EV Table 1.E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey's post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.
Figure 5A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01
Figure 6A. Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months. Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining. The contrast of photomicrographs was inverted.B. Dendritic spines from heterozygous hTauATmice, seen by Golgi staining.C. Quantification of dendritic spine densities in control littermate and hTauAT mice. Note that the spine density in heterozygous hTauAT is increased (control littermates: n = 2 animals; heterozygous hTauATn = 4 animals; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal; unpaired t-test).D. Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12±1 month old mice demonstrating significantly enhanced basal synaptic transmission (Multiple repeat ANOVA,F(9, 126)=2.00, p=0.044) in area CA3 in mice expressing hTauAT. Scale bars: 500 µV vertical bar and 20 msec horizontal bar.E. Long-term potentiation in the mossy fiber pathway demonstrates no differences in long-term plasticity between control (black) and hTauAT expressing mice (red).F. Input / output curve of field potential recording in stratum pyramidale (s.p.) of area CA3 in organotypic hippocampal slices at DIV 30. Somatic field potentials are increased in hTauAT compared to control littermates. Scale bars: 800 µV vertical bar and 20 msec horizontal bar.
Figure 7A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).
Figure 8A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey's post-hoc test.D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).
Figure 1A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testB. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testC. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test
Figure 4A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison testF. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test
Figure 5A. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed. Data information: Statistical significance was assessed using log-rank test.B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not affect the expression of any of the innate immunity genes (C, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.
Figure 6A. To examine the role of the ATFS-1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo-6 mutants, we examined the effect of disrupting atfs-1 in nuo-6 mutants. Loss of atfs-1 completely abolishes the increased resistance to bacterial pathogens in nuo-6 worms. p-value indicates significance of difference between red and purple lines. nuo-6; atfs-1 contains the gk3094 deletion allele. Three biological replicates per strain were measured. B. To examine the role of ATFS-1 in the upregulation of innate immunity genes in nuo-6 mutants, we compared gene expression between nuo-6 and nuo-6; atfs-1 mutants with wild-type worms and atfs-1 mutants as controls. Deletion of atfs-1 significantly decreases the expression of genes involved in innate immunity in nuo-6 worms but does not decrease the expression of these genes in wild-type worms. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Worms in a wild-type background (control) are shown with white bars, while worms with atfs-1 deletion are shown with blue bars. Data information: Error bars indicate SEM. **p<0.01, ***p<0.001. Statistical significance in panel A was assessed using log-rank test. Statistical significance in panel B was assessed using a two-way ANOVA with Bonferroni posttest.
Figure 7A. Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs-1 mutants (et15 and et17) revealed that innate immunity genes are upregulated when ATFS-1 is activated. This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38-mediated innate immune pathway. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test.
Figure 1Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum. Eluates were analysed by SDS-PAGE and western botting. Total 1.5 %, Eluate, 100 %.Wild type mitochondria were solubilized and subjected to 2D-BN/SDS-PAGE and western blotting with indicated antibodies.Solubilized mitochondria from MITRAC12FLAG- or ACAD9FLAG-expressing cells were subjected to anti-FLAG immunoisolation and native elution. Eluates were analysed by 2D-BN/SDS-PAGE and western blot analysis. Total 0.8 % (MITRAC12FLAG isolation), 0.35 % (ACAD9FLAG isolation), Eluate 100 %.Solubilized mitochondria from MITRAC15FLAG-expressing cells were first subjected to native anti-FLAG immunoisolation (Total 0.1 %, Eluate 10 %, lane 4). Purified complexes were applied to anti-MITRAC12 immunoisolation (Eluates 100 %, lane 5). All samples were analyzed by SDS-PAGE and immunoblotting.
Figure 2Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %.Solubilized mitochondria from MITRAC15FLAG- or TIM21FLAG-expressing cells were subjected to anti-FLAG immunoisolation and analyzed by 2D-BN/SDS-PAGE and western blotting. MITRAC15FLAG isolation: Total 0.1 %, Eluate 100 %. TIM21FLAG isolation: Total 0.2 %, Eluate 66 % (TIM21FLAG decoration), 33 % (MITRAC15, ACAD9, MITRAC12, COX1 decoration).Solubilized mitochondria from MITRAC15FLAG- or C12ORF62FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.4 %, Eluate 100 % (MITRAC15FLAG isolation), 70 % (C12ORF62FLAG isolation).Solubilized mitochondria from ACAD9FLAG-or MITRAC15FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.5 % (ACAD9FLAG isolation), 0.4 % (MITRAC15FLAG isolation) Eluate 100 %. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.