Article Figures & Data
Figures
- Figure 1.
17βHSD10 is expressed and active at a comparable level in astrocytes from the mouse cortex, hippocampus, and cerebellum. A, Representative confocal images of astrocytes immunostained for 17βHSD10 (in cyan) and mitochondrial dye MitoTracker Red CMXRos (in magenta) confirm the mitochondrial localization of the protein. Scale bars: 10 μm. B, Representative immunoblot showing the protein expression levels of 17βHSD10 and mitochondrial COXIV in primary astrocytes with β-actin used as a loading control. C, Quantification of Western blot analysis indicating that both COXIV (F(2,12) = 0.93, p = 0.42, n.s.) and 17βHSD10 (F(2,12) = 0.85, p = 0.45, n.s.) maintain comparable levels of expression in astrocytes from the cortex (CX), hippocampus (HI), and cerebellum (CE) and normalizing 17βHSD10 to COXIV also showed no differences between astrocytes (F(2,12) = 0.28, p = 0.76, n.s.). Bar graphs represent the mean ± SEM; n = 5 independent primary cultures; analysis through one-way ANOVA with Tukey’s post hoc comparisons. D, Representative confocal images of live astrocytes treated with 20 μm CHANA for 30 min. CHANA is broken down by 17βHSD10 to its fluorescent product CHANK which can be detected by measuring fluorescence levels in the sample (shown in yellow). Scale bar: 10 μm. E, CHANK fluorescence measured over 30 min showed increased fluorescence from baseline in all groups (F(1,45) = 88.87, p < 0.001) with no differences between astrocytes from the three brain regions (F(2,45) = 1.60, p = 0.17, n.s; interaction F(2,15) = 0.56, p = 0.58, n.s.). Analysis through mixed ANOVA with Tukey’s post hoc comparisons; n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: (-)-cyclohexenyl amino naphthalene alcohol (CHANA), cyclohexenyl amino naphthalene ketone (CHANK), cortex (CX), hippocampus (HI), and cerebellum (CM). Extended Data Figures 1-1, 1-2, 1-3, and 1-4 further show that 17βHSD10 is expressed in uncultured astrocytes of both neonatal and adult mice.
- Figure 2.
Overexpressing 17βHSD10 in cortical astrocytes induces elongated mitochondrial networks, regardless of 17βHSD10 enzymatic activity. A, 17βHSD10 expression levels were assessed through Western blot analysis 10 d after lentiviral transduction. A representative immunoblot of three independent primary culture preparations and the quantification of 17βHSD10 in these cells showed that the lentiviral protocol induced 20-fold increase in protein expression with both the mutant and wt versions of 17βHSD10 as compared with control astrocytes (F(2,18) = 16.67, p < 0.001). Bar graphs represent the mean ± SEM. Analysis through one-way ANOVA with Tukey’s post hoc comparisons on n = 6 independent culture preparations. B, 17βHSD10 enzymatic activity as measured by CHANA turnover rate was significantly increased only when the wt variant of 17βHSD10 was overexpressed, while the mutated version did not cause significant increase (F(2,24) = 13.24, p < 0.001). Bar graphs represent the mean ± SEM. Analysis through one-way ANOVA with Tukey’s post hoc comparisons on n = 9 independent culture preparations. C, Representative confocal microscopy images of astrocytes immunostained for 17βHSD10 and mitochondrial dye MitoTracker Red CMXRos confirmed mitochondrial expression of the enzyme in both control and overexpressed conditions. Scale bars: 10 μm. The arrows with dotted lines in the control image indicate predominant mitochondrial morphology in normal conditions; arrows with solid lines in mut-17βHSD10 and wt-17βHSD10 panels indicate abnormal elongated and highly branched network morphology. D, Mitochondrial footprint was uniform across conditions (F(2,751) = 0.308, p = 0.75, n.s.). E, Mean branch length was greater in mut-17βHSD10 and wt-17βHSD10 populations (F(2,712) = 30.16, p < 0.004). F, Total branch length increased in the overexpression conditions as compared with control (F(2,715) = 212.56, p < 0.001) and (G) network branching (F(2,715) = 1.95, p = 0.256) remained unchanged. Bar graphs represent the mean ± SEM. Analysis via mixed model analysis with Tukey’s post hoc comparisons; n = 3 independent primary culture preparations with 244–254 cells analyzed per condition; n.s, non-significant, ***p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 3.
Increased 17βHSD10 activity decreased mitochondrial respiration, while ETC inhibition decreased the activity of the enzyme and AG18051 countered these effects. A, A schematic representation of the ETC targets and utilized inhibitors used for the mitochondrial bioenergetic test. B, Timescale of the experiment, inhibitor administration, and measurement parameters utilized in the respiratory test. C, Mitochondrial respiration was assessed by measuring OCR which was normalized to the protein content in each sample. This profile was further used to calculate the respiratory parameters in the next panel. D, Overexpression of mut-17βHSD10 did not affect the respiratory parameters (ps > 0.05); however, wt-17βHSD10 overexpression reduced maximal respiration (F(3,40) = 14.22, p < 0.001), as well as spare respiratory capacity (F(3,40) = 26.737, p < 0.001) in cortical astrocytes, while basal respiration (F(3,40) = 2.04, p = 0.12, n.s.), ATP production (F(3,40) = 1.93, p = 0.14, n.s.), and proton leak (F(3,40) = 1.43, p = 0.25, n.s.) remained unaffected. These effects were recovered to baseline when cells were pretreated with 17βHSD10 inhibitor AG18051 (20 μm; ps > 0.05). E, Similarly, while all cells with normal activity levels of 17βHSD10 were able to upregulate respiration when challenged, high activity of the protein reduced this metabolic compensation (F(3,40) = 13.89, p < 0.001). F, Both endogenous and overexpressed 17βHSD10 activity was inhibited by OM, AA, and Rot (control: F(4,30) = 29.99, p < 0.001; mut-17βHSD10: F(4,25) = 13.73, p < 0.001 and wt-17βHSD10 (F(4,25) = 12.09, p < 0.001), while mitochondrial decoupling with FCCP did not affect 17βHSD10 activity (ps > 0.05). Graphs represent the mean ± SEM. Compounds: FCCP (carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone: 4 μm), OM (1.5 μm), AA (5 μm), Rot (5 μm). Treatment time: acute injection. AG18051 (20 μm) was administered for 24 h before the experiment. One-way between-subjects ANOVA with Tukey’s post hoc comparisons (n = 4–6 independent primary culture preparations); n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A (AA), oligomycin (OM), rotenone (ROT). Extended Data Figure 3-1 shows the effects of AG18051 on respiratory function of astrocytes with endogenous 17βHSD10 levels.
- Figure 4.
17βHSD10 expression and activity increased following ischemia-reoxygenation (IR) insult, affecting superoxide generation and mitochondrial network morphology. A, Western blot analysis of mitochondrial protein expression showed (B) that 17βHSD10 (F(2,15) = 7.61, p = 0.005) was increased following IR in the absence of glucose, with VDAC1 following similar pattern (F(2,15) = 18.94, p < 0.001), while COXIV remained stable (F(2,12) = 0.88, p = 0.44, n.s.). C, IR reduced viability (treatment main effect: F(1,66) = 72.12, p < 0.001) to a similar level in all astrocytes (17βHSD10 expression main effect: F(2,66) = 0.06, p = 0.94, n.s.). D, Cytotoxicity was increased by the insult (treatment main effect: F(1,66) = 24.15, p < 0.001) with all three groups showing similar change (17βHSD10 expression main effect: F(2,66) = 0.32, p = 0.73, n.s.). Analysis via two-way between subjects ANOVA with Tukey’s post hoc test, n = 4 independent biological replicates; n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ischemia-reoxygenation (IR), Voltage Dependent Anion Channel 1 (VDAC1), Cytochrome c oxidase (COX). Treatment: hypoxia, 6 h (0 mm glucose 0.5% O2; 5% CO2 at 37°C) followed by 2 h of reoxygenation.
- Figure 5.
17βHSD10 activity increased following IR and this affected superoxide generation and mitochondrial network morphology. A, 17βHSD10 activity increased following IR in all astrocytes and AG18051 inhibited this elevation (F(4,45) = 20.88, p < 0.001); n = 3 independent primary culture preparations. Analysis via two-way between subjects ANOVA with Tukey’s post hoc test. B, Superoxide generation was increased following the insult and the effects were ameliorated by AG18051 in all astrocytes but mut-17βHSD10-overexpressing cells. The increase was of higher magnitude in wt-17βHSD10-overexpressing cells, and the AG18051-mediated decrease was particularly pronounced in this condition (interaction: F(4,27) = 3.18, p = 0.029). Analysis on n = 3 independent biological replicates using two-way between subjects ANOVA with Tukey’s post hoc test. C, AG18051 did not rescued the cytotoxic effect induced by IR only in astrocytes overexpressing wt-17βHSD10 (interaction: F(4,111) = 6.03, p < 0.001). D, Representative confocal images of mitochondrial networks in astrocytes in control and IR conditions. Arrows with dotted line indicate fragmented mitochondrial morphology with reduced branching, while arrows with solid line show elongated and highly branched network morphology. Scale bars: 10 μm. E, Mitochondrial footprint changes following IR depended on 17βHSD10 expression and catalytic activity (interaction between treatment and 17βHSD10 expression phenotype: F(2,243) = 96.80, p < 0.001) with substantial reduction in normal astrocytes, nonsignificant effect in mut-17βHSD10-overexpressing cells and an increase in the wt-17βHSD10-overexpressing group. F, Average branch length decreased following IR and this was significant in 17βHSD10-overexpressing astrocytes (main effect of 17βHSD10 expression: F(2,243) = 14.29, p = 0.015). G, Total branch length changes following IR depended on 17βHSD10 expression and catalytic activity (interaction between treatment and 17βHSD10 expression phenotype: F(2,261) = 7.77, p = 0.041) with only wt-17βHSD10-overexpressing astrocytes resisting the decrease in this parameter following IR insult. H, Network branching also showed significant interaction between treatment and 17βHSD10 expression phenotype (F(2,243) = 22.14, p = 0.007), whereby wt-17βHSD10 showed an increase of branch number following treatment. Analysis via two-way between subjects ANOVA with Tukey’s post hoc test, n = 3 independent biological replicates, 45 cells per condition; n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ischemia-reoxygenation (IR). Treatment: hypoxia, 6 h (0 mm glucose 0.5% O2; 5% CO2 at 37°C) followed by 2 h of reoxygenation.
- Figure 6.
Oligomeric Aβ(1-42) increased 17βHSD10 expression in cortical astrocytes, while overexpression of wt-17βHSD10 exacerbated Aβ(1-42)-induced cell death. A, Representative immunoblot showing alterations in mitochondrial protein expression following a 48-h treatment with 0–1 μm oligomeric Aβ(1-42). B, Quantitative analysis showed that oligomeric Aβ(1-42) elevated 17βHSD10 expression (F(2,12) = 5.65, p = 0.019), while the other two proteins were not affected (COXIV: F(2,12) = 0.22, p = 0.81, n.s.; VDAC1: F(2,12) = 0.67, p = 0.53, n.s.). Analysis via one-way between subjects ANOVA with Tukey’s post hoc test on n = 3 independent primary culture preparations. C, Viability was significantly reduced only in wt-17βHSD10-overexpressing astrocytes treated with Aβ(1-42) (interaction between 17βHSD10 expression and treatment F(2,48) = 3.36, p = 0.044). D, Cytotoxicity was elevated by Aβ(1-42) in all astrocytes (main effect of treatment: F(1,48) = 80.08, p < 0.001) and wt-17βHSD10 expression particularly potentiated this increase (main effect of 17βHSD10 expression: F(2,48) = 5.95, p = 0.005); two-way between subjects ANOVA on n = 4 biological replicates. Graphs shows mean ± SEM; n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ischemia-reoxygenation (IR), Voltage Dependent Anion Channel 1 (VDAC1), Cytochrome c oxidase (COX).
- Figure 7.
The catalytic activity of overexpressed 17βHSD10 decreased following treatment with oligomeric Aβ(1-42), and this was associated with exacerbated superoxide generation and mitochondrial fragmentation. A, Oligomeric Aβ(1-42) reduced detected 17βHSD10 activity only in wt-17βHSD10-overexpressing astrocytes, and AG18051 co-administration abolished this difference (interaction between treatment and 17βHSD10 expression: F(4,63) = 178.45, p < 0.001). B, Oligomeric Aβ(1-42) increased superoxide generation in all astrocytes and the effect was of greatest magnitude in the wt-17βHSD10-overexpressing group, while AG18051 countered the increase in all conditions (F(2,63) = 10.34, p < 0.001). Bar graphs show mean ± SEM; n = 3 independent primary cultures; two-way between subjects ANOVA with Tukey’s post hoc comparisons. C, AG18051 did not rescue the cytotoxic effect induced by Aβ(1-42) (ps > 0.05). D, Representative confocal images of astrocytic mitochondrial networks in control and Aβ(1-42) treatment. Arrows with dotted line indicate fragmented mitochondrial morphology which was particularly pronounced in wt-17βHSD10-overexpressing astrocytes. Scale bars: 10 μm. E, Mitochondrial footprint was reduced in all astrocytes following amyloid treatment (treatment main effect: F(1,702) = 30.79, p = 0.031) in all three groups of astrocytes (F(2,702) = 0.50, p = 0.640). F, Changes in mean branch length depended on both 17βHSD10 expression and amyloid treatment (interaction F(2,702) = 21.17, p = 0.007). G, Total branch length also displayed a significant interaction (F(2,702) = 12.58, p = 0.019) whereby amyloid reduced summed branch length only in cells overexpressing the catalytically active form of the protein. H, Network branching also showed significant interaction between treatment and protein expression (F(2,702) = 13.59, p = 0.016) with only wt-17βHSD10-overexpressing cells showing significant decrease in the number of mitochondrial branches following amyloid treatment. Analysis via two-way between subjects ANOVA with Tukey’s post hoc test, n = 3 biological replicates with 120 cells analyzed per treatment; n.s, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Treatments: Aβ(1-42) 1 μm for 48 h; AG18051 20 μm.
Tables
Figure Test p-value Shapiro–Wilk Sample N Mean SEM Figure 1C One-way ANOVA with Tukey’s post hoc test 0.452 0.126 17βHSD10-cortex 5 1.094 0.096 0.949 17βHSD10 hippocampus 5 1.084 0.060 0.858 17βHSD10-cerebellum 5 1.221 0.089 One-way ANOVA with Tukey’s post hoc test 0.420 <0.001 COXIV-cortex 5 1.096 0.091 0.992 COXIV-hippocampus 5 1.002 0.084 0.098 COXIV-cerebellum 5 1.153 0.059 One-way ANOVA with Tukey’s post hoc test 0.760 0.833 17βHSD10/COXIV-cortex 5 1.061 0.137 0.454 17βHSD10/COXIV-hippocampus 5 1.203 0.100 0.311 17βHSD10/COXIV-cerebellum 5 1.143 0.160 Figure 1E Mixed ANOVA 0.849 cortex-0 min 6 4972.333 26.911 Time (main) <0.001 0.049 cortex-10 min 6 5023.167 34.622 Region (main) 0.583 0.610 cortex-20 min 6 5173.000 37.605 interaction 0.170 0.159 cortex-30 min 6 5297.000 50.440 0.584 hippocampus-0 min 6 4974.333 26.911 0.096 hippocampus-10 min 6 5007.167 34.622 0.407 hippocampus-20 min 6 5127.500 37.605 0.059 hippocampus-30 min 6 5186.333 50.440 0.119 cerebellum-0 min 6 4969.167 26.911 0.332 cerebellum-10 min 6 5069.000 34.622 0.552 cerebellum-20 min 6 5176.500 37.605 0.562 cerebellum-30 min 6 5252.167 50.440 Figure 2A One-way ANOVA with Tukey’s post hoc test <0.001 0.042 control 7 1.191 0.316 0.258 mut-17βHSD10 7 18.429 2.879 0.078 wt-17βHSD10 7 18.573 3.096 Figure 2B One-way ANOVA with Tukey’s post hoc test <0.001 0.244 control 9 113.119 1.098 0.781 mut-17βHSD10 9 115.079 1.570 0.018 wt-17βHSD10 9 439.268 89.354 Figure 2D Mixed linear model with Tukey’s post hoc test <0.001 control 254 685.867 17.373 <0.001 mut-17βHSD10 244 703.992 20.494 <0.001 wt-17βHSD10 254 684.278 15.635 Main effect of expression F(2,743) = 0.308, p = 0.691, ηp2 = 0.133 Main effect of culture replicate F(2,743) = 0.029, p = 0.971, ηp2 = 0.014 Interaction F(4,743) = 1.331, p = 0.257, ηp2 = 0.007 Figure 2E Mixed linear model with Tukey’s post hoc test <0.001 control 255 1.761 0.021 <0.001 mut-17βHSD10 235 2.049 0.033 <0.001 wt-17βHSD10 223 2.002 0.030 Main effect of expression F(2,704) = 30.155, p = 0.004, ηp2 = 0.938 Main effect of culture replicate F(2,704) = 0.211, p = 0.818, ηp2 = 0.095 Interaction F(4,704) = 1.075, p = 0.368, ηp2 = 0.006 Figure 2F Mixed linear model with Tukey’s post hoc test <0.001 0.001 control 253 2.929 0.058 <0.001 mut-17βHSD10 246 4.172 0.078 <0.001 wt-17βHSD10 225 3.971 0.079 Main effect of expression F(2,715) = 212.558, p < 0.001, ηp2 = 0.991 Main effect of culture replicate F(2,715) = 0.449, p = 0.667, ηp2 = 0.182 Interaction F(4,715) = 0.417, p = 0.797, ηp2 = 0.002 Figure 2G Mixed linear model with Tukey’s post hoc test 0.009 <0.001 control 255 2.677 0.095 <0.001 mut-17βHSD10 246 3.070 0.111 <0.001 wt-17βHSD10 203 3.078 0.119 Main effect of expression F(2,715) = 8.267, p = 0.256, ηp2 = 0.494 Main effect of culture replicate F(2,715) = 0.254, p = 0.787, ηp2 = 0.112 Interaction F(4,715) = 2.535, p = 0.0.39, ηp2 = 0.014 Figure 3B One-way ANOVA with Tukey’s post hoc test 0.124 Basal respiration 0.196 control 11 9459.894 702.399 0.319 mut-17βHSD10 11 9365.145 461.574 0.207 wt-17βHSD10 11 8227.585 634.004 0.525 wt-17βHSD10-AG18051 11 10,289.873 550.445 One-way ANOVA with Tukey’s post hoc test <0.001 Maximal respiration 0.524 control 11 16,182.764 849.316 0.566 mut-17βHSD10 11 15,564.403 692.724 0.663 wt-17βHSD10 11 10,644.094 931.124 0.749 wt-17βHSD10-AG18051 11 17,685.873 740.995 One-way ANOVA with Tukey’s post hoc test <0.001 Spare capacity 0.471 control 11 6722.869 287.169 0.854 mut-17βHSD10 11 6199.257 407.046 0.675 wt-17βHSD10 11 2416.509 485.998 0.902 wt-17βHSD10-AG18051 11 7396.000 511.162 One-way ANOVA with Tukey’s post hoc test <0.001 ATP 0.205 control 11 7116.955 510.235 0.987 mut-17βHSD10 11 6894.442 377.057 0.295 wt-17βHSD10 11 6103.095 494.693 0.398 wt-17βHSD10-AG18051 11 7620.969 426.466 One-way ANOVA with Tukey’s post hoc test 0.248 Proton leak 0.322 control 11 2342.939 218.751 0.047 mut-17βHSD10 11 2470.702 236.700 0.026 wt-17βHSD10 11 2124.490 171.068 0.210 wt-17βHSD10-AG18051 11 2767.481 261.637 Figure 3C One-way ANOVA with Tukey’s post hoc test <0.001 mut-17βHSD10 0.680 control 6 105.412 2.603 0.268 FCCP 6 99.763 3.576 0.868 OM 6 100.262 9.700 0.385 AA 6 69.928 3.920 0.563 ROT 6 63.441 3.079 One-way ANOVA with Tukey’s post hoc test 0.239 mut-17βHSD10-AG18051 0.601 control 5 96.463 7.560 0.576 FCCP 5 97.961 6.000 0.523 OM 5 86.966 7.697 0.046 AA 5 98.821 10.812 0.213 ROT 5 77.290 3.938 One-way ANOVA with Tukey’s post hoc test <0.001 wt-17βHSD10 0.219 control 6 174.683 3.501 0.079 FCCP 6 197.215 7.434 0.435 OM 6 185.665 28.768 0.158 AA 6 114.067 7.088 0.858 ROT 6 87.097 4.562 One-way ANOVA with Tukey’s post hoc test 0.168 wt-17βHSD10-AG18051 0.827 control 4 99.756 10.407 0.745 FCCP 5 102.113 4.069 0.552 OM 5 92.224 6.437 0.126 AA 5 102.260 10.680 0.086 ROT 5 78.412 5.266 Figure 3D One-way ANOVA with Tukey’s post hoc test <0.001 0.205 control 11 174.205 5.353 0.897 mut-17βHSD10 11 167.062 5.353 0.495 wt-17βHSD10 11 129.654 5.353 0.451 wt-17βHSD10-AG18051 7 166.592 6.710 Extended Data
Fig. 3-1BTwo-tailed unpaired t tests 0.337 Basal respiration
Control
AG1805111
69460
10,712702.4
11370.036 Maximal respiration
Control
AG1805111
616,183
19,850849.3
15160.005 Spare capacity
Control
AG1805111
66723
9138287.2
868.80.741 ATP production
Control
AG1805111
67117
7435510.2
875.70.023 Proton leak
Control
AG1805111
62343
3372218.8
381.3Figure 4B One-way ANOVA with Tukey’s post hoc test 0.005 17βHSD10 0.898 Control 6 0.925 0.133 0.066 IR 6 1.431 0.121 0.176 IR+glucose 6 0.852 0.083 One-way ANOVA with Tukey’s post hoc test <0.001 VDAC1 0.873 Control 6 0.850 0.089 0.592 IR 6 1.840 0.172 0.203 IR+glucose 6 0.891 0.110 One-way ANOVA with Tukey’s post hoc test 0.442 17βHSD10/COXIV 0.753 Control 5 0.864 0.099 0.005 IR 5 0.766 0.098 0.055 IR+glucose 5 0.693 0.076 Figure 4C Mixed ANOVA 0.314 control-control 12 100.000 3.410 Group (main) 0.290 0.112 control-IR 12 82.999 4.985 Treatment (main) <0.001 0.716 mut-17βHSD10-control 12 103.521 4.272 interaction 0.977 0.533 mut-17βHSD10-IR 12 86.108 2.443 0.145 wt-17βHSD10-control 12 101.045 3.106 0.271 wt-17βHSD10-IR 12 85.337 5.805 Figure 4D Mixed ANOVA 0.026 control-control 12 2.221 0.243 Group (main) 0.002 0.020 control-IR 12 15.781 3.006 Treatment (main) 0.522 0.050 mut-17βHSD10-control 12 1.563 0.212 interaction <0.001 0.189 mut-17βHSD10-IR 12 15.269 3.001 0.500 wt-17βHSD10-control 12 2.043 0.116 0.088 wt-17βHSD10-IR 12 16.045 2.333 Figure 5A Mixed ANOVA Group (main) <0.001 0.244 control-control 6 127.860 9.436 Treatment (main) <0.001 0.901 control-IR 6 323.533 4.313 interaction <0.001 0.582 control-IR-AG18051 6 173.563 4.331 0.141 mut-17βHSD10-control 6 140.961 9.189 0.114 mut-17βHSD10-IR 6 283.245 7.154 0.031 mut-17βHSD10-IR-AG18051 6 215.737 22.617 0.655 wt-17βHSD10-control 6 361.384 26.606 0.011 wt-17βHSD10-IR 6 1026.559 133.923 0.070 wt-17βHSD10-IR-AG18051 6 196.246 18.773 Figure 5B Mixed ANOVA 0.475 control-control 6 96.693 6.355 Group (main) 0.093 0.029 control-IR 3 160.736 8.422 Treatment (main) <0.001 0.830 control-IR-AG18051 3 112.557 21.545 interaction 0.029 0.385 mut-17βHSD10-control 6 96.662 16.852 0.322 mut-17βHSD10-IR 3 150.052 36.172 0.179 mut-17βHSD10-IR-AG18051 3 137.171 21.279 0.153 wt-17βHSD10-control 6 85.644 9.051 0.776 wt-17βHSD10-IR 3 240.813 12.960 0.673 wt-17βHSD10-IR-AG18051 3 133.154 18.287 Figure 5C Two-way ANOVA control-control 7 2.879 0.393 Group (main) <0.001 control-IR 7 8.036 0.840 Treatment (main) 0.444 control-IR-AG18051 7 8.943 1.399 interaction <0.001 mut-17βHSD10-control 21 2.658 0.503 mut-17βHSD10-IR 21 8.764 0.960 mut-17βHSD10-IR-AG18051 21 6.591 1.647 wt-17βHSD10-control 12 1.875 0.369 wt-17βHSD10-IR 12 11.429 0.834 wt-17βHSD10-IR-AG18051 12 2.905 0.495 Figure 5E Mixed linear model with Tukey’s post hoc test <0.001 control-control 45 640.292 34.194 <0.006 control-IR 45 254.570 15.413 0.022 mut-17βHSD10-control 45 719.772 35.778 0.039 mut-17βHSD10-IR 45 647.206 35.228 0.012 wt-17βHSD10-control 45 694.020 34.624 0.051 wt-17βHSD10-IR 45 903.350 39.936 Main effect of expression F(1,243) = 22.702, p = 0.006 Main effect of treatment F(1,243) = 38.381, p = 0.022 Interaction F(2,243) = 96.796, p < 0.001 Main effect of culture replicate F(2,243) = 0.674, p = 0.568 Figure 5F Mixed linear model with Tukey’s post hoc test <0.001 control-control 45 1.420 0.025 0.331 control-IR 45 1.394 0.017 0.941 mut-17βHSD10-control 45 1.633 0.012 0.412 mut-17βHSD10-IR 45 1.542 0.024 0.126 wt-17βHSD10-control 45 1.774 0.025 <0.001 wt-17βHSD10-IR 45 1.685 0.016 Main effect of expression F(1,243) = 14.491, p = 0.015 Main effect of treatment F(1,243) = 15.029, p = 0.060 Interaction F(2,243) = 0.190, p = 0.834 Main effect of culture replicate F(2,243) = 0.399, p = 0.941 Figure 5G Mixed linear model with Tukey’s post hoc test 0.018 control-control 45 1.949 0.074 0.308 control-IR 45 1.563 0.065 0.046 mut-17βHSD10-control 45 4.152 0.144 0.003 mut-17βHSD10-IR 45 2.661 0.158 0.005 wt-17βHSD10-control 45 4.350 0.204 0.128 wt-17βHSD10-IR 45 4.501 0.121 Main effect of expression F(1,243) = 65.562, p = 0.001 Main effect of treatment F(1,243) = 8.388, p = 0.101 Interaction F(2,243) = 7.770, p = 0.041 Main effect of culture replicate F(2,243) = 0.612, p = 0.629 Figure 5H Mixed linear model with Tukey’s post hoc test 0.689 control-control 45 1.352 0.036 0.772 control-IR 45 1.309 0.025 0.062 mut-17βHSD10-control 45 2.530 0.077 0.003 mut-17βHSD10-IR 45 1.684 0.081 <0.001 wt-17βHSD10-control 45 2.443 0.108 0.158 wt-17βHSD10-IR 45 2.682 0.076 Main effect of expression F(1,243) = 45.037, p = 0.002 Main effect of treatment F(1,243) = 6.758, p = 0.121 Interaction F(2,243) = 22.136, p = 0.007 Main effect of culture replicate F(2,243) = 0.232, p = 0.805 One-way ANOVA with Tukey’s post hoc test 0.019 17βHSD10 Figure 6B 0.900 0 nm 5 0.972 0.113 0.650 100 nm 5 1.317 0.081 0.819 1000 nm 5 1.366 0.072 One-way ANOVA with Tukey’s post hoc test 0.809 VDAC1 <0.001 0 nm 3 1.067 0.067 0.949 100 nm 3 1.029 0.141 0.902 1000 nm 3 1.155 0.182 One-way ANOVA with Tukey’s post hoc test 0.528 17βHSD10/COXIV 0.463 0 nm 5 0.944 0.067 0.683 100 nm 5 0.775 0.061 0.202 1000 nm 5 0.851 0.154 Figure 6C Mixed ANOVA 0.304 control-control 8 109.015 4.408 Group (main) 0.002 0.815 control-Aβ 8 115.155 5.896 Treatment (main) 0.165 0.209 mut-17βHSD10-control 8 112.757 5.435 interaction 0.044 0.229 mut-17βHSD10-Aβ 8 109.097 4.470 0.508 wt-17βHSD10-control 8 104.241 6.616 0.034 wt-17βHSD10-Aβ 8 83.870 3.612 Figure 6D Mixed ANOVA 0.216 control-control 8 0.873 0.385 Group (main) 0.005 0.003 control-Aβ 8 3.202 0.397 Treatment (main) <0.001 0.395 mut-17βHSD10-control 8 0.942 0.334 interaction 0.285 0.761 mut-17βHSD10-Aβ 8 3.248 0.375 0.698 wt-17βHSD10-control 8 1.485 0.302 0.076 wt-17βHSD10-Aβ 8 4.815 0.379 Figure 7A Mixed ANOVA Group (main) <0.001 0.796 control-control 8 97.837 3.182 Treatment (main) <0.001 0.870 control-Aβ 8 98.334 2.863 interaction <0.001 0.454 control-Aβ-AG18051 8 104.191 3.936 0.147 mut-17βHSD10-control 8 96.317 2.262 0.827 mut-17βHSD10-Aβ 8 91.433 4.324 0.859 mut-17βHSD10-Aβ-AG18051 8 95.197 4.067 0.713 wt-17βHSD10-control 8 374.482 11.633 0.444 wt-17βHSD10-Aβ 8 324.850 11.826 0.168 wt-17βHSD10-Aβ-AG18051 8 104.093 2.924 Figure 7B Mixed ANOVA 0.641 control-control 8 100.000 10.408 Group (main) 0.242 0.400 control-Aβ 8 171.792 24.816 Treatment (main) <0.001 0.901 control-Aβ-AG18051 8 103.411 16.303 interaction 0.204 0.108 mut-17βHSD10-control 8 106.125 10.044 0.150 mut-17βHSD10-Aβ 8 172.123 22.324 0.788 mut-17βHSD10-Aβ-AG18051 8 78.465 16.038 0.003 wt-17βHSD10-control 8 114.667 13.606 0.296 wt-17βHSD10-Aβ 8 241.006 39.597 0.111 wt-17βHSD10-Aβ-AG18051 8 81.276 7.345 Figure 7C Two-way ANOVA control-control 8 0.873 0.385 control-Aβ 8 4.024 0.182 Group (main) 0.017 control-Aβ-AG18051 8 3.558 0.589 Treatment (main) <0.001 mut-17βHSD10-control 8 0.942 0.333 interaction 0.521 mut-17βHSD10-Aβ 8 3.274 0.375 mut-17βHSD10-Aβ-AG18051 8 2.590 0.413 wt-17βHSD10-control 8 1.784 0.266 wt-17βHSD10-Aβ 8 4.606 0.447 wt-17βHSD10-Aβ-AG18051 8 3.284 0.474 Figure 7E Mixed linear model with Tukey’s post hoc test 0.048 control-control 120 738.426 26.021 0.006 control-Aβ 120 559.937 22.975 <0.001 mut-17βHSD10-control 120 796.907 32.100 <0.001 mut-17βHSD10-Aβ 120 536.263 25.395 <0.001 wt-17βHSD10-control 120 743.826 22.845 0.231 wt-17βHSD10-Aβ 120 476.186 15.730 Main effect of expression F(2,702) = 0.500, p = 0.640 Main effect of treatment F(1,702) = 30.749, p = 0.031 Interaction F(2,702) = 0.668, p = 0.562 Main effect of culture replicate F(2,702) = 1.113, p = 0.454 Figure 7F Mixed linear model with Tukey’s post hoc test 0.006 control-control 120 2.004 0.024 <0.001 control-Aβ 120 1.804 0.034 0.007 mut-17βHSD10-control 120 2.439 0.036 <0.001 mut-17βHSD10-Aβ 120 1.933 0.040 <0.001 wt-17βHSD10-control 120 2.324 0.034 <0.001 wt-17βHSD10-Aβ 120 1.681 0.013 Main effect of expression F(2,702) = 3.629, p = 0.034 Main effect of treatment F(1,702) = 78.927, p = 0.012 Interaction F(2,702) = 21.179, p = 0.007 Main effect of culture replicate F(2,702) = 2.323, p = 0.226 Figure 7G Mixed linear model 0.011 control-control 120 3.194 0.084 <0.001 0.399 control-Aβ 120 3.127 0.081 <0.001 <0.001 mut-17βHSD10-control 120 4.382 0.117 <0.001 0.066 mut-17βHSD10-Aβ 120 4.463 0.111 <0.001 wt-17βHSD10-control 120 4.080 0.110 <0.001 wt-17βHSD10-Aβ 120 2.753 0.150 Main effect of expression F(2,702) = 5.364, p = 0.074 Main effect of treatment F(1,702) = 22.354, p = 0.042 Interaction F(2,702) = 12.575, p = 0.019 Main effect of culture replicate F(2,702) = 0.526, p = 0.640 Figure 7H Mixed linear model <0.001 control-control 120 3.311 0.127 <0.001 control-Aβ 120 3.148 0.110 <0.001 mut-17βHSD10-control 120 3.709 0.201 <0.001 mut-17βHSD10-Aβ 120 4.070 0.187 <0.001 wt-17βHSD10-control 120 4.130 0.189 <0.001 wt-17βHSD10-Aβ 120 3.111 0.189 Main effect of expression F(2,702) = 2.047, p = 0.244 Main effect of treatment F(1,702) = 207.886, p = 0.005 Interaction F(2,702) = 13.591, p = 0.016 Main effect of culture replicate F(2,702) = 3.066, p = 0.216
Extended Data Figure 1-1
17βHSD10 is expressed in cortical (CX), hippocampal (HI), and cerebellar (CM) astrocytes of neonatal and adult mice. Low-magnification images of acute brain slices stained for 17βHSD10 (magenta) and astrocytic marker GFAP (cyan) in the frontal cortex, hippocampus, and cerebellar cortex. Colocalization is indicated in white. Colocalization analysis quantified using Manders’ coefficient showing the fraction of GFAP staining overlapping with 17βHSD10 signal is high in cortical (2 d old: M = 0.969; 7 d old: M = 0.901; 2 months old: M = 0.753; 4 months old: M = 0.958), hippocampal (2 d old: M = 0.825; 7 d old: M = 0.711; 2 months old: M = 0.847; 4 months old: M = 0.764), and cerebellar (2 d old: M = 0.954; 7 d old: M = 0.795; 2 months old: M = 0.605; 4 months old: M = 0.648) astrocytes. Scale bar: 300 μm. Download Figure 1-1, EPS file.
Extended Data Figure 1-2
Mouse cortical astrocytes express 17βHSD10. High-magnification images of acute brain slices stained for 17βHSD10 (magenta) and GFAP (cyan). The first column presents the overlay of GFAP and 17βHSD10 staining with colocalization shown in white. 17βHSD10 and GFAP staining alone are observed in the following two columns. The fourth column shows the outlines of the GFAP staining obtained in ImageJ through enhancing (0.25%) and equalizing the contrast of the image followed by the “find edges” function. The final column shows the overlays of the GFAP outlines and 17βHSD10 staining, facilitating the representation of the colocalization. Scale bar: 30 μm. Download Figure 1-2, EPS file.
Extended Data Figure 3-1
17βHSD10 inhibition with AG18051 caused upregulated maximal respiration, space respiratory capacity, and proton leak. A, OCR was normalized to the protein content in each sample. This profile was further used to calculate the respiratory parameters in the next panel. B, Although basal respiration and overall ATP production remained unaffected by AG18051 (t(15) = 0.99, p = 0.338 and t(15) = 0.33, p = 0.741, respectively), maximal respiration (t(15) = 2.30, p = 0.036), and spare respiratory capacity (t(15) = 3.27, p = 0.005) was increased in astrocytes treated with the inhibitor. Treatment time: acute injection. AG18051 (20 μm) was administered for 24 h prior to the experiment. Independent samples t tests (n = 4–6 independent primary culture preparations); *p < 0.05, **p < 0.01, ***p < 0.001. Download Figure 3-1, EPS file.
Extended Data Figure 1-4
Mouse cerebellar astrocytes express 17βHSD10. High-magnification images of acute brain slices stained for 17βHSD10 (magenta) and GFAP (cyan) with colocalization shown in white. The first column presents the overlay of GFAP and 17βHSD10 staining. 17βHSD10 and GFAP staining alone are shown in the following two columns. The fourth column shows the outlines of the GFAP staining obtained in ImageJ through enhancing (0.25%) and equalizing the contrast of the image followed by the “find edges” function. The final column shows the overlays of the GFAP outlines and 17βHSD10 staining, facilitating the representation of the colocalization. Scale bar: 30 μm. Download Figure 1-4, EPS file.
Extended Data Figure 1-3
Mouse hippocampal astrocytes express 17βHSD10. High-magnification images of acute brain slices stained for 17βHSD10 (magenta) and GFAP (cyan) with colocalization shown in white. The first column presents the overlay of GFAP and 17βHSD10 staining. 17βHSD10 and GFAP staining alone are shown in the following two columns. The fourth column shows the outlines of the GFAP staining obtained in ImageJ through enhancing (0.25%) and equalizing the contrast of the image followed by the “find edges” function. The final column shows the overlays of the GFAP outlines and 17βHSD10 staining, facilitating the representation of the colocalization. Scale bar: 30 μm. Download Figure 1-3, EPS file.
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