H pCRP * / mCRP dependent NETosis in isolated human neutrophils detected by confocal immunofluorescence microscopy . Isolated neutrophils incubated for 3 hours at 37 ° C , 5 % CO2 with 100 µg / ml pCRP with and without PC : LPC liposomes ( LP ) and C10M ( molar ratio 1 : 100 , pCRP : C10M ) , respectively . Control was left unstimulated and 100 nM phorbol 12 - myristate 13 - acetate ( PMA ) served as positive control . Cells were washed , fixed and stained , and analyzed by confocal microscopy . Results are given as a ratio of NETing cells / all cells per ROI ,

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H pCRP*/mCRP dependent NETosis in isolated human neutrophils detected by confocal immunofluorescence microscopy. Isolated neutrophils incubated for 3 hours at 37 °C, 5% CO2 with 100 µg/ml pCRP with and without PC:LPC liposomes (LP) and C10M (molar ratio 1:100, pCRP:C10M), respectively. Control was left unstimulated and 100 nM phorbol 12-myristate 13-acetate (PMA) served as positive control. Cells were washed, fixed and stained, and analyzed by confocal microscopy. Results are given as a ratio of NETing cells/all cells per ROI,


B Detection of C10M by mass spectrometry in rat urine . Renal excretion of C10M detected by mass spectrometry in three rats ( biological replicates , n = 3 ) intravenously injected with C10M . Urine samples were collected at the indicated timepoints . 80 % of the applied C10M mass was excreted after 90

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B Detection of C10M by mass spectrometry in rat urine. Renal excretion of C10M detected by mass spectrometry in three rats (biological replicates, n=3) intravenously injected with C10M. Urine samples were collected at the indicated timepoints. 80% of the applied C10M mass was excreted after 90 min


C Immunohistochemistry of rat kidneys subjected to IRI and i . p . pCRP application revealed distinct staining by anti - pCRP * / mCRP - 9C9 antibody ( green , arrows ) . C10M reduces the deposition of total CRP in the impaired tissue . No deposits in the non - ischemic tissue ( sham ) . Exemplary stainings out of at least three are shown .

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C Immunohistochemistry of rat kidneys subjected to IRI and i.p. pCRP application revealed distinct staining by anti-pCRP*/mCRP-9C9 antibody (green, arrows). C10M reduces the deposition of total CRP in the impaired tissue. No deposits in the non-ischemic tissue (sham). Exemplary stainings out of at least three are shown.


D Tissue lysates of rat kidney were separated on SDS - PAGE and total CRP was identified with anti - CRP antibody . A band at the size of mCRP ( ~ 23 kDa ) was detected in kidneys subjected to IRI and pCRP , but not in animals treated additionally with C10M . The household gene protein GAPDH served as a control for loading equal amounts of protein . 50 and 100 ng human pCRP , respectively , served as a positive control . Representative results are shown for replicated assays ( n = 3 ) .

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D Tissue lysates of rat kidney were separated on SDS-PAGE and total CRP was identified with anti-CRP antibody. A band at the size of mCRP (~23 kDa) was detected in kidneys subjected to IRI and pCRP, but not in animals treated additionally with C10M. The household gene protein GAPDH served as a control for loading equal amounts of protein. 50 and 100 ng human pCRP, respectively, served as a positive control. Representative results are shown for replicated assays (n=3).


E Renal excretion is impaired by pCRP * / mCRP - driven tissue damage . Blood urea nitrogen ( BUN ) was utilized as surrogate marker for the excretion function of the kidney . Blood samples were taken before the surgical procedure ( preoperative ) and 24 hours after the procedure ( harvest ) . Graph shows mean ± SEM .

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E Renal excretion is impaired by pCRP*/mCRP-driven tissue damage. Blood urea nitrogen (BUN) was utilized as surrogate marker for the excretion function of the kidney. Blood samples were taken before the surgical procedure (preoperative) and 24 hours after the procedure (harvest). Graph shows mean ± SEM.


F Immunohistochemical detection of transmigrated CD68 + cells in IRI kidneys . Quantification of immunohistochemical results is shown as mean ± SEM . pCRP ( 25 µg / ml ) increased the number of CD68 + cells transmigrated into injured renal tissue significantly , while C10M abolished these effects . Presented are mean cell counts per ROI in each animal .

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F Immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. Quantification of immunohistochemical results is shown as mean ± SEM. pCRP (25 µg/ml) increased the number of CD68+ cells transmigrated into injured renal tissue significantly, while C10M abolished these effects. Presented are mean cell counts per ROI in each animal.


G Periodic acid - Schiff ( PAS ) stained kidney sections show increased damage after renal IRI in rats when pCRP ( 25 µg / ml ) was injected i . v . The tubulointerstitial injury was quantified by the loss of tubular brush border and by cast formation following an established protocol ( Megyesi et al , 1998 , 2001 ) . Quantification of immunohistochemical results is shown as mean ± SEM .

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G Periodic acid-Schiff (PAS) stained kidney sections show increased damage after renal IRI in rats when pCRP (25 µg/ml) was injected i.v. The tubulointerstitial injury was quantified by the loss of tubular brush border and by cast formation following an established protocol (Megyesi et al, 1998, 2001). Quantification of immunohistochemical results is shown as mean ± SEM.


H Representative results for the immunohistochemical detection of transmigrated CD68 + cells in IRI kidneys . CD68 + cells are stained with HistoGreen substrate ( green ) . Scale bars indicate 100 µm .

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H Representative results for the immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. CD68+ cells are stained with HistoGreen substrate (green). Scale bars indicate 100 µm.


I Representative results for the periodic acid - Schiff ( PAS ) stained kidney sections quantified in ( G ) . Scale bars indicate 100 µm .

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I Representative results for the periodic acid-Schiff (PAS) stained kidney sections quantified in (G). Scale bars indicate 100 µm.


B Kaplan - Meier plots for control , pCRP , and pCRP + C10M ( above ) and C10M vs control ( bottom ) treatment for total allograft survival with and without CRP ± C10M . Kaplan - Meier curves for different treatments were compared by Mantel - Cox log - rank test . Allograft survival was significantly reduced by pCRP administration ( P = 0 . 0005 , median survival control vs pCRP , 7 . 8 vs 4 . 8 days ) . C10M masks the CRP - accelerated hindlimb rejection ( median survival 7 . 2 days ) . Biological replicates , n = 4 per group .

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B Kaplan-Meier plots for control, pCRP, and pCRP+C10M (above) and C10M vs control (bottom) treatment for total allograft survival with and without CRP ± C10M. Kaplan-Meier curves for different treatments were compared by Mantel-Cox log-rank test. Allograft survival was significantly reduced by pCRP administration (P=0.0005, median survival control vs pCRP, 7.8 vs 4.8 days). C10M masks the CRP-accelerated hindlimb rejection (median survival 7.2 days). Biological replicates, n=4 per group.


C Representative photographic examples of transplanted VCA hindlimb allografts . Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation . Rats receiving i . p . pCRP presented VCA with massive edema ( left ) . C10M treatment inhibits pCRP - aggravated early graft rejection ( middle ) . The depicted C10M control without CRP shows no clinical signs of rejection after five days ( right ) .

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C Representative photographic examples of transplanted VCA hindlimb allografts. Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation. Rats receiving i.p. pCRP presented VCA with massive edema (left). C10M treatment inhibits pCRP-aggravated early graft rejection (middle). The depicted C10M control without CRP shows no clinical signs of rejection after five days (right).


D , E Representative immunofluorescence images from transplanted grafts reveal distinct signals ( green ) using anti - CD68 and anti - CRP antibodies , respectively . In C10M treated animals , less CD68 + cells infiltrate the transplanted tissue . Scale bars , 100 μm . ( D ) Immunofluorescence of transplanted muscle and ( E ) skin tissue revealed distinct CRP - deposition ( green ) in pCRP treated animals , but only minimal staining in rats treated additionally with C10M .

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D,E Representative immunofluorescence images from transplanted grafts reveal distinct signals (green) using anti-CD68 and anti-CRP antibodies, respectively. In C10M treated animals, less CD68+ cells infiltrate the transplanted tissue. Scale bars, 100 μm. (D) Immunofluorescence of transplanted muscle and (E) skin tissue revealed distinct CRP-deposition (green) in pCRP treated animals, but only minimal staining in rats treated additionally with C10M.


F Exemplary tissue lysates of muscle and skin probes separated on SDS - PAGE are depicted . Total human CRP was identified with an anti - CRP antibody .

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F Exemplary tissue lysates of muscle and skin probes separated on SDS-PAGE are depicted. Total human CRP was identified with an anti-CRP antibody.


A Phagocytosis of pCRP - opsonized , heat - killed and FITC - labeled S . pneumoniae by human monocytes serves as exemplary flow cytometry - based phagocytosis assay . Bar chart shows phagocytic index ( percentage of target positive cells of subtype / all cells of subtype ) of un - opsonized ( control , dark - grey ) and pCRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively . B Phagocytosis of pCRP - opsonized , heat - killed and FITC - labeled E . coli by human monocytes serves as a second exemplary flow cytometry - based phagocytosis assay . Bar chart shows phagocytic index as described above of un - opsonized ( control , dark - grey ) and pCRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively . C Phagocytosis of pCRP - opsonized , heat - treated and FITC - labeled zymosan by human monocytes serves as a third exemplary flow cytometry - based phagocytosis assay . Bar chart shows phagocytic index as described above of un - opsonized ( control , dark - grey ) and CRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively .

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A Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human monocytes serves as exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index (percentage of target positive cells of subtype / all cells of subtype) of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. B Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human monocytes serves as a second exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. C Phagocytosis of pCRP-opsonized, heat-treated and FITC-labeled zymosan by human monocytes serves as a third exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and CRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.


D Experiments described in ( A ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) and C10M ( 1 : 100 molar ratio ) for 30 min , 37 ° C . Bar chart shows phagocytic index of S . pneumoniae - positive monocytes for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively . E Experiments described in ( B ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) and C10M ( 1 : 100 molar ratio ) for 30 min , 37 ° C . Bar chart shows phagocytic index of E . coli - positive monocytes for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively . F Experiments described in ( C ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) and C10M ( 1 : 100 molar ratio ) for 30 min , 37 ° C . Bar chart shows phagocytic index of zymosan - positive monocytes for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively .

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D Experiments described in (A) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of S. pneumoniae-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. E Experiments described in (B) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of E. coli-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. F Experiments described in (C) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of zymosan-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.


G Phagocytosis of pCRP - opsonized , heat - killed and FITC - labeled S . pneumoniae by human neutrophils . The same blood samples described in ( A ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of un - opsonized ( control , dark - grey ) and pCRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively . H Phagocytosis of pCRP - opsonized , heat - killed and FITC - labeled E . coli by human neutrophils . The same blood samples described in ( B ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of un - opsonized ( control , dark - grey ) and pCRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively . I Phagocytosis of pCRP - opsonized , heat - killed and FITC - labeled zymosan by human neutrophils . The same blood samples described in ( C ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils . Bar chart shows phagocytic index of un - opsonized ( control , dark - grey ) and pCRP - opsonized targets ( red ) after 5 , 10 , 15 , and 20 min , respectively .

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G Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human neutrophils. The same blood samples described in (A) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. H Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human neutrophils. The same blood samples described in (B) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. I Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled zymosan by human neutrophils. The same blood samples described in (C) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.


J Phagocytosis of pCRP + C10M - treated S . pneumoniae by human neutrophils . Results from experiments described in ( D ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of S . pneumoniae - positive neutrophils for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively . K Phagocytosis of pCRP + C10M - treated E . coli by human neutrophils . Results from experiments described in ( E ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of E . coli - positive neutrophils for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively . L Phagocytosis of pCRP + C10M - treated zymosan by human neutrophils . Results from experiments described in ( F ) were analyzed for the phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of zymosan - positive neutrophils for un - opsonized ( control , dark - grey ) and targets incubated with pCRP and C10M ( green ) after 5 , 10 , 15 , and 20 min , respectively .

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J Phagocytosis of pCRP+C10M-treated S. pneumoniae by human neutrophils. Results from experiments described in (D) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of S. pneumoniae-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. K Phagocytosis of pCRP+C10M-treated E. coli by human neutrophils. Results from experiments described in (E) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of E. coli-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. L Phagocytosis of pCRP+C10M-treated zymosan by human neutrophils. Results from experiments described in (F) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of zymosan-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.


B . 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 .

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B. 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 .

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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 ) .

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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 .

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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 .

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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 ) .

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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 ) .

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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 .

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I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.


A . Conformational change of Tau detected by ALZ - 50 antibody immunoreactivity in stratum lucidum ( asterisks ) of area CA3 and somata in stratum pyramidale ( arrowheads ) .

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A. 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 .

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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 ) .

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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 ) .

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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).


A . 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 .

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A. 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 ) .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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


A . 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 ) .

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A. 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 .

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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 ) .

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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 .

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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.


A . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A. 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 ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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).


A . 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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<0.001


C . 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 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 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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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).


A . 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

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 test


B . 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

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. 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 test


C . 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 -

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C. 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


A - D . Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay . Both nuo - 6 and isp - 1 long - lived mitochondrial mutants show increased resistance compared to wild - type worms ( A ) . Disruption of components of the p38 - mediated innate immune signaling pathway ( nsy - 1 , sek - 1 , pmk - 1 , atf - 7 ) markedly decreases resistance to PA14 in wild - type ( B ) , nuo - 6 ( C ) and isp - 1 ( D ) worms . All strains were tested in a single parallel experiment . Two biological replicates per strain were performed with a total of at least 175 animals per strain . Data information : * * * p < 0 . 001 . Statistical significance for survival plots was determined with the log - rank test . Significance is indicated between the strain listed on top and all other strains . Data from panel A is repeated in panels B , C , and D for direct comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared to wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C) and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. Data information: ***p<0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A is repeated in panels B, C, and D for direct comparison.


A - H . To examine the role of the p38 - mediated innate immune signaling pathway in the long - lifespan of the nuo - 6 and isp - 1 mitochondrial mutants , we crossed the long - lived mitochondrial mutants to worms with mutations in nsy - 1 , sek - 1 , pmk - 1 and atf - 7 ( gof ) . In each case , mutation of genes involved in p38 - mediated innate immunity signaling markedly reduces the lifespan of the long - lived mitochondrial mutant but have little or no impact on wild - type lifespan . In panel G , we utilized pmk - 1 RNAi instead of the pmk - 1 mutation because of the close proximity of isp - 1 and pmk - 1 on the same chromosome . EV indicates empty vector . The lifespan results for panels A , B , D , E , F , and H were performed in a single parallel experiment . Results are from a minimum of three biological replicates per strain . Data information : Statistical significance for survival plots was determined with the log - rank test . p - values indicate significance between red and purple lines . Control strains are shown in multiple panels for direct comparison .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain. Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.


A , 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

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,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 test


F . 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

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. 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


A . 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 .

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A. 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 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A . 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 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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.


A . 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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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.


Solubilized 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 % .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Solubilized 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 .

O O O O O O O O O O O O O O O O O O O O O O

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 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O

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 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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.


Mitochondrial 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 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondrial 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 ) .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O

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 ) .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O

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 .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O

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.


Mitochondria from wild type and MITRAC15 - / - cells were isolated and subjected to steady - state protein analyses by western blotting .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting.


Mitochondria from wild type and MITRAC15 - / - cells were isolated and analyzed by BN - PAGE . Triton X - 100 ( left site ) or digitonin ( right site ) were used for solubilization . The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels ( error bars indicate SEM from three biological replicates ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).


Complex I and citrate synthase activity were measured in WT and MITRAC15 - / - cells ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).


Pulse - chase [ 35S ] methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).


Pulse radiolabeling after re - expressing MITRAC15FLAG in MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Relative pulse signal intensity of ND2 was normalized to ATP6 ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 ; unpaired t - test ) .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).


Mitochondrial translation products were radiolabelled with [ 35S ] methionine after siRNA - mediated depletion of ACAD9 in wild type and MITRAC15 - / - cells . Cell lysates were analyzed by SDS - PAGE and digital autoradiography . For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal . ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 * * * p < 0 . 001 ; unpaired t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).


FLAG - immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG - expressing cells after siRNA - mediated down regulation of ACAD9 and pulse radiolabeling . SDS - PAGE followed by autoradiography was used to analyze eluates . Relative signal intensity of ND2 , ND3 and ND4L were normalized to ATP6 ( total ) or isolation efficiency ( eluate ) . Total 2 % , Eluate 100 % . Error bars indicate SEM from three biological replicates ; * * p < 0 . 01 ; unpaired t - test .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FLAG-immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG-expressing cells after siRNA-mediated down regulation of ACAD9 and pulse radiolabeling. SDS-PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3 and ND4L were normalized to ATP6 (total) or isolation efficiency (eluate). Total 2 %, Eluate 100 %. Error bars indicate SEM from three biological replicates; **p < 0.01; unpaired t-test.