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Mitochondrial spongiotic brain disease: astrocytic stress and harmful rapamycin and ketosis effect

View ORCID ProfileOlesia Ignatenko, View ORCID ProfileJoni Nikkanen, View ORCID ProfileAlexander Kononov, Nicola Zamboni, View ORCID ProfileGulayse Ince-Dunn, View ORCID ProfileAnu Suomalainen  Correspondence email
Olesia Ignatenko
1Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
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  • ORCID record for Olesia Ignatenko
Joni Nikkanen
2Cardiovascular Research Institute, University of California, San Francisco, CA, USA
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Alexander Kononov
3Cancer Research UK, University of Manchester, Manchester, UK
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Nicola Zamboni
4Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland
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Gulayse Ince-Dunn
1Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
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  • ORCID record for Gulayse Ince-Dunn
Anu Suomalainen
1Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
5Neuroscience Center, University of Helsinki, Helsinki, Finland
6HUSlab, Helsinki University Hospital, Helsinki, Finland
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  • For correspondence: anu.wartiovaara@helsinki.fi
Published 31 July 2020. DOI: 10.26508/lsa.202000797
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  • Figure 1.
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    Figure 1. ISRmt is induced in the brain in response to loss of mtDNA helicase Twinkle in astrocytes.

    (A) Top: schematic of molecular events upon Twnk knockout. Bottom: mouse models used in the study (Ignatenko et al, 2018). (B) mRNA expression of ISRmt genes measured by RT-quantitative PCR. TwKOastro n = 8 and their controls n = 6–7; TwKOneuro n = 8–9 and their controls n = 11. (C) Immunoblot analysis of transsulfuration pathway enzymes, CTH and CBS. n = 6 per group. (D) GFD15 mRNA expression measured by RT-quantitative PCR, A.U.; “1” indicates detectable expression, N.D. indicates no detectable expression. TwKOastro n = 8 and their controls n = 7, TwKOneuro n = 5, and their controls n = 5. (E) Amino acids, metabolomics. TwKOastro n = 8 and their controls n = 8, TwKOneuro n = 5, and their controls n = 6. Multiplicity corrected P-values are shown, calculated using the Benjamini–Hochberg procedure. (F) Immunoblot analysis of S6 and phospho-S6. n = 6 per group. (G) ISRmt is induced in TwKOastro brain, schematic representation. Ctr, control mice. For all immunoblots, densitometric quantification of signals is plotted and band intensities are normalized to total protein on the membrane. (B, C, F) P-values are shown, calculated using unpaired two-tailed parametric t test. For all graphs, FC is a fold change ratio, relative to corresponding Ctr mice. For all box and whiskers plots, the box extends from the 25th to 75th percentiles; whiskers show all points minimum to maximum; symbols indicate biological replicates. Results of statistical testing can be found in Table S4 and Supplemental Data 1 and 2.

  • Figure S1.
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    Figure S1. Cell-specific loss of mtDNA helicase Twinkle causes differential responses in the mouse brain.

    (A) Body weight, preterminal stage. TwKOastro n = 8 and their controls n = 7, TwKOneuro n = 5, and their controls n = 5. (B) mtDNA amount, measured using quantitative PCR to DNA and normalized to nuclear DNA amount. TwKOastro n = 8 and their controls n = 7; TwKOneuro preterminal n = 5 and their controls n = 4; TwKOneuro preterminal n = 8 and their controls n = 7. (C) mRNA expression of ISRmt genes measured by RT-quantitative PCR. TwKOneuro n = 8 and their controls n = 7. (D) Amino acids, metabolomics. TwKOastro n = 8 and their controls n = 8; TwKOneuro n = 5 and their controls n = 6. (E) Amino acids, metabolomics. TwKOneuro n = 8 and their controls n = 8. (F) Purine precursors and degradation products, and uracil, metabolomics. TwKOastro n = 8 and their controls n = 8, TwKOneuro preterminal n = 5 and their controls n = 6; TwKOneuro young n = 8 and their controls n = 8. For all graphs, FC is a fold change ratio, relative to corresponding Ctr mice. For all box and whiskers plots, the box extends from the 25th to 75th percentiles; whiskers show all points minimum to maximum; symbols indicate biological replicates. (A, B, C) P-values are shown, calculated using unpaired two-tailed parametric test. (D, E, F) Multiplicity corrected P-values are shown, calculated using the Benjamini–Hochberg procedure. Results of statistical testing can be found in Table S4 and Supplemental Data 1 and 2.

  • Figure 2.
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    Figure 2. Rapamycin does not prevent or rescue brain pathology of TwKOastro.

    (A, B, C, D, E, F, G, H, I) Rapamycin treatment started at presymptomatic (A, B, C, D, E, F) and at a moderately advanced (G, H, I) phenotype stage. (A) Schematic of presymptomatic rapamycin treatment. Daily i.p. injections of rapamycin or vehicle were initiated at 1.5 mo and were continued until humane end point in body weight loss was reached (2.3 mo, arrow). Green area shows treatment period and pink area represents symptom progression in untreated TwKOastro mice. (B) Body weight progression upon rapamycin treatment initiated at the presymptomatic stage. Weight change is shown as a fold change (FC) relative to the body weight at treatment initiation (zero time point). n = 9 per group. Mean with SD is shown. (C) Immunoblot analysis of total S6 and phospho-S6 proteins. Ctr Veh n = 4, Ctr Rapa n = 4, TwKOastro Rapa n = 4, TwKOastro Veh n = 5. 2× is a Ctr Veh sample, with a twofold amount of protein loaded on the lane compared with other samples. (D) Brain pathology, rapamycin treatment. Left: Representative cortical sections show spongiotic encephalopathy. Hematoxylin and eosin staining of TwKOastro treated with vehicle or rapamycin. Arrows show sponge-like holes typical for spongiotic encephalopathy. Quantification below: counts of holes per field of view, n = 7 mice per group, two sections per mouse, three fields of view per section. Counts from the fields of view of one mouse are plotted with the same symbol. Scale bars, 50 μm. Right: Astrocyte activation. Glial fibrillary acidic protein (GFAP), immunofluorescent detection, cortex, representative of n = 7 mice per group, two sections per mouse. Below: mRNA expression of GFAP and vimentin (reactive gliosis markers) measured by RT-quantitative PCR. Ctr Veh n = 8, Ctr Rapa n = 8, TwKOastro Veh n = 7–8, TwKOastro Rapa n = 7. (E) Metabolomics analysis. All metabolites with P-value < 0.01 in any of the two-way ANOVA tests (interaction, genotype, and treatment) are plotted. Z-scores are calculated from intensity values. Note that metabolite is plotted more than once if it passes selection criteria in more than one test. n = 6 per group. Full data set is shown in Fig S2C. (F) Metabolomics, full data set is shown in Fig S2C. n = 6 per group. Multiplicity corrected P-values are shown, calculated using the Benjamini–Hochberg procedure. (G, H, I) Rapamycin treatment started at a moderately advanced phenotype stage. (G) Schematic of the rapamycin treatment at a moderately advanced disease stage of TwKOastro mice. Daily i.p. injections of rapamycin or vehicle were started at a moderately advanced phenotype stage (3 mo) and were continued until humane end point in body weight loss was reached (3.5 mo, arrow). Green area shows treatment period and the pink area represents symptom progression in untreated TwKOastro mice. (H) Body weight progression upon rapamycin treatment initiated at a moderately advanced disease stage. Ctr Veh n = 3, Ctr Rapa n = 5, TwKOastro Veh n = 7, TwKOastro Rapa n = 8. Weight change is shown as an FC relative to the body weight at treatment initiation (zero time point). Mean with SD is shown. (I) Quantification of spongiotic encephalopathy. Counts of holes per field of view in brain sections stained with hematoxylin and eosin, 1–2 sections per mouse, three fields of view per section. Counts from fields of view of the same mouse are plotted with the same symbol. TwKOastro Veh n = 6, TwKOastro Rapa n = 8. Ctr, control mice; Veh, vehicle-treated mice; Rapa, rapamycin-treated mice. (C, D) P-values are shown, calculated using two-way ANOVA followed by Tukey correction for multiple comparisons. For all graphs, FC is a fold change ratio, relative to corresponding Ctr mice. For all box and whiskers plots, the box extends from the 25th to 75th percentiles; whiskers show all points minimum to maximum; symbols indicate biological replicates. Results of statistical testing can be found in Table S4 and Supplemental Data 2.

  • Figure S2.
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    Figure S2. Rapamycin treatment started at presymptomatic age.

    (A) mRNA expression of ISRmt genes measured by RT-quantitative PCR. Ctr Veh n = 8, Ctr Rapa n = 8, TwKOastro Veh n = 8, TwKOastro Rapa n = 7. P-values are shown, calculated using two-way ANOVA followed by Tukey correction for multiple comparisons. (B) Immunoblot analysis of transsulfuration pathway enzymes CTH and CBS. Densitometry of Western blot signals: a protein signal normalized to the total protein in the membrane. Ctr Veh n = 4, Ctr Rapa n = 4, TwKOastro Rapa n = 4. TwKOastro Veh n = 5. 2x is a Ctr Chow sample with a twofold amount of protein compared with other samples. P-values are shown, calculated using two-way ANOVA followed by Tukey correction for multiple comparisons. (C) Metabolomics analysis, all identified metabolites, Z-scores are calculated from intensity values. n = 6 per group. (D) Amino acids, metabolomics. n = 6 per group. P-values are shown, calculated using unpaired two-tailed parametric t test. For all graphs, FC is a fold change ratio, relative to corresponding Ctr mice. For all box and whiskers plots, the box extends from the 25th to 75th percentiles; whiskers show all points minimum to maximum; symbols indicate biological replicates. Results of statistical testing can be found in Table S4 and Supplemental Data 2.

  • Figure 3.
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    Figure 3. Ketogenic diet accelerates brain pathology of TwKOastro.

    Ketogenic diet started upon weaning of the mice. (A) Schematic representation of the ketogenic diet experiment. Ketogenic or chow diet was administered to the mice upon weaning. Experiment was continued until humane end point body weight loss of TwKOastro was reached (3.2 mo, arrow). Green area shows a treatment period and pink area represents a symptom progression in untreated TwKOastro mice. (B) Blood β-hydroxybutyrate levels 8 wk after start of the experiment. Ctr Chow n = 10, Ctr Keto n = 7, TwKOastro Chow n = 9, TwKOastro Keto n = 8. (C) Body weight progression. Ctr Chow n = 12, Ctr Keto n = 8, TwKOastro Chow n = 16, TwKOastro Keto n = 14. Weight change is shown as fold change (FC) relative to the zero time point. Mean with SD is shown. (D) Brain pathology, ketogenic diet. Left: Spongiotic encephalopathy. Representative cortical sections, hematoxylin and eosin staining of TwKOastro fed with chow or ketogenic diet. Arrows show sponge-like holes typical for spongiotic encephalopathy. Quantification below: counts of holes per field of view, TwKOastro Chow n = 6, TwKOastro Keto = 8, one to two sections per mouse, three fields of view per section. Counts from the fields of view of one mouse are plotted with the same symbol. Scale bars, 50 μm. Right: Astrogliosis. GFAP, immunofluorescent detection, cortex, representative of n = 8 mice per group, two sections per mouse. Below: mRNA expression of GFAP and vimentin (astrocyte activation markers) measured using RT-quantitative PCR. Ctr Chow n = 7, Ctr Keto n = 8, TwKOastro Chow n = 8, TwKOastro Keto n = 8. (E) Immunoblot analysis of transsulfuration pathway enzymes CTH and CBS. Densitometry of Western blot signals: a protein signal normalized to the total protein in the membrane. n = 5 per group. 2× is a Ctr Chow sample with a twofold amount of protein loaded on the lane, compared with other samples. (F) Metabolomics analysis. Metabolites with P-value < 0.01 in the two-way ANOVA interaction tests, and a cluster of metabolites with P-value < 0.01 in the two-way ANOVA genotypes tests are plotted (see selection of the cluster in Fig S3D). Z-scores are calculated from intensity values. Note that metabolite is plotted more than once if it passes selection criteria in more than one test. Ctr Chow, Ctr Keto n = 6; TwKOastro Chow, TwKOastro Keto n = 5. Full data set is shown in Fig S3C. (G) Metabolomics, full data set is shown in Fig S3C. Ctr Chow n = 6, Ctr Keto n = 6, TwKOastro Chow n = 5, TwKOastro Keto n = 5. Multiplicity corrected P-values are shown, calculated using the Benjamini–Hochberg procedure. Ctr, control mice; Chow, mice fed with chow diet; Keto, mice fed with ketogenic diet. For all graphs, FC is a fold change ratio, relative to corresponding Ctr mice. For all box and whiskers plots, the box extends from the 25th to 75th percentiles; whiskers show all points minimum to maximum; symbols indicate biological replicates. (C, D, E) P-values are shown, calculated using two-way ANOVA followed by Tukey correction for multiple comparisons. Results of statistical testing can be found in Table S4 and Supplemental Data 2.

  • Figure S3.
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    Figure S3. Ketogenic treatment started at weaning.

    (A) mRNA expression of ISRmt genes measured by RT-quantitative PCR. Ctr Chow n = 7, Ctr Keto n = 8, TwKOastro Chow n = 8, TwKOastro Keto n = 8. P-values are shown, calculated using two-way ANOVA followed by Tukey correction for multiple comparisons. (B) GFD15 mRNA is detected only in TwKOastro mice, measured by RTquantitative PCR to cDNA synthesized from total tissue RNA. Ratio of TwKOastro on ketogenic diet to TwKOastro on chow diet. n = 8 per group. P-value is shown, calculated using unpaired two-tailed parametric t test. (C) Metabolomics analysis, all identified metabolites, Z-scores are calculated from intensity values. Ctr Chow n = 6, Ctr Keto n = 6, TwKOastro Chow n = 5, TwKOastro Keto n = 5. (D) Metabolomics analysis. Metabolites with P-value < 0.01 two-way ANOVA genotype and treatment tests are shown. Cluster 1 outlined in green is shown on Fig 3F. Z-scores are calculated from intensity values. Note that metabolite is plotted more than once if it passes selection criteria in more than one test. Ctr Chow n = 6, Ctr Keto n = 6, TwKOastro Chow n = 5, TwKOastro Keto n = 5. Full data set is shown in Fig S3C. (E) Amino acids, metabolomics. Ctr Chow n = 6, Ctr Keto n = 6, TwKOastro Chow n = 5, TwKOastro Keto n = 5. Multiplicity corrected P-values are shown, calculated using the Benjamini–Hochberg procedure. Graphs (A, B, E): FC is a fold change ratio relative to corresponding Ctr Chow mice; box and whiskers plots, box extends from the 25th to 75th percentiles, whiskers show all points minimum to maximum, symbols indicate biological replicates. Results of statistical testing can be found in Table S4 and Supplemental Data 2.

  • Figure S4.
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    Figure S4. Complete Western blot membranes shown in the article figures. Dashed lines indicate the cropped area.

Supplementary Materials

  • Figures
  • Supplemental Data 1.

    Targeted metabolomics and statistical analysis.[LSA-2020-00797_Supplemental_Data_1.xlsx]

  • Supplemental Data 2.

    Untargeted metabolomics and statistical analysis.[LSA-2020-00797_Supplemental_Data_2.xlsx]

  • Table S1 Mouse cohorts used in the study.

  • Table S2 Primer sequences used in the study.

  • Table S3 Antibodies used in the study.

  • Table S4 Results of statistical analyses of data presented in the figures.

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Treating astrocyte mtDNA depletion
Olesia Ignatenko, Joni Nikkanen, Alexander Kononov, Nicola Zamboni, Gulayse Ince-Dunn, Anu Suomalainen
Life Science Alliance Jul 2020, 3 (9) e202000797; DOI: 10.26508/lsa.202000797

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Treating astrocyte mtDNA depletion
Olesia Ignatenko, Joni Nikkanen, Alexander Kononov, Nicola Zamboni, Gulayse Ince-Dunn, Anu Suomalainen
Life Science Alliance Jul 2020, 3 (9) e202000797; DOI: 10.26508/lsa.202000797
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Volume 3, No. 9
September 2020
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