The Autophagic Activator GHF-201 Can Alleviate Pathology in a Mouse Model and in Patient Fibroblasts of Type III Glycogenosis
<p>Weight over time in WT mice, <span class="html-italic">Agl<sup>−/−</sup></span> (KO) mice injected intramuscularly biweekly with 10% DMSO solvent control, and KO mice injected in the same way with 250 mg/kg GHF-201. n = 8 WT/Veh, n = 6 KO/Veh, and n = 10 KO/GHF-201 mice were used. Throughout the period, KO mice were heavier than WT mice (<span class="html-italic">p</span> < 0.0001, Two Way ANOVA with repeated measures). Due to a >10% weight reduction between biweekly weightings, one mouse was removed from the KO/Veh group on day 116 after treatment initiation and one mouse was removed from the KO/GHF201 group on day 52 after treatment initiation.</p> "> Figure 2
<p>Movement distance (<b>A</b>), sojourn in the center of an open field arena (inset representative open field sojourn heatmaps) (<b>B</b>), grip strength (<b>C</b>), and latency to fall from a rotating rod (<b>D</b>) were measured in n = 8 <span class="html-italic">Agl<sup>−/−</sup></span> mice treated with 10% DMSO vehicle (KO/Veh), n = 6 wild type mice treated with vehicle (WT/Veh), and n = 10 <span class="html-italic">Agl<sup>−/−</sup></span> mice treated with GHF-201 (KO/GHF-201). In (<b>D</b>), each rotarod session included three consecutive runs (1–3), separated by 10 min pauses. Two Way ANOVA with repeated measures shows that only in (<b>C</b>) GHF-201 treatment values were higher than vehicle-treated values in KO mice throughout the period (<span class="html-italic">p</span> < 0.05). Linear regression analysis of overall latency over time (<b>D</b>), shows that WT animals developed a significantly higher learning capacity than KO/Veh and KO/GHF-201 mice. Training capacity, shown by increase in latency over runs, was only demonstrated in WT mice 80 days post-treatment initiation. See text for statistical analysis. *, KO/GH201 is significantly different from KO/Veh; *#, KO/GHF-201 is significantly different from both KO/Veh and WT/Veh; *^, KO/GHF-201 is significantly different only from KO/Veh and not from WT/Veh (correction effect). Difference significance determined by multiple <span class="html-italic">t</span>-tests with Sidak post hoc correction. All error bars represent s.e.m.</p> "> Figure 3
<p>Blood metabolic panel based on n = 8 <span class="html-italic">Agl<sup>−/−</sup></span> mice treated with 10% DMSO vehicle (KO/Veh), n = 6 wild type mice treated with vehicle (WT/Veh), and n = 10 <span class="html-italic">Agl<sup>−/−</sup></span> mice treated with GHF-201, as indicated. At all time points, GHF-201 significantly reduced alanine transferase and creatine phosphokinase, demonstrating partial restoration of liver and muscle damages, respectively. Additionally, blood glucose was increased, and blood triglycerides were decreased by GHF-201 in treated <span class="html-italic">Agl<sup>−/−</sup></span> mice. Statistical significance between KO/Veh and KO/GHF-201 at the different time points was determined by Two Way ANOVA with repeated measures with a Tukey post hoc test according to simple effects within rows (i.e., times). *, 0.01 < <span class="html-italic">p</span> < 0.05; **, 0.001 < <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>Indirect calorimetry and metabolic cage analysis. Following a 48 h habituation period, mice (n = 4 from each study arm indicated) were monitored over a 24 h period. Data are mean ± SEM from eight-month-old mice. ((<b>A</b>)—Respirometry) As demonstrated by the positive mean differences, in the light, dark, and overall (total), vehicle-treated <span class="html-italic">Agl<sup>−/−</sup></span> mice demonstrate lower respiratory exchange ratio (RER, (<b>A1</b>)), total energy expenditure (TEE, (<b>A2</b>–<b>A4</b>)), and carbohydrate oxidation (<b>A6</b>). As shown by the negative mean differences, fat oxidation was reduced in <span class="html-italic">Agl<sup>−/−</sup></span> mice in light, dark, and overall as compared to WT mice (<b>A5</b>). All these parameters were increased or corrected by GHF-201 at all times, except for fat oxidation in the dark and overall (<b>A4</b>). ANCOVA results suggest that weight-dependent TEE is reduced in <span class="html-italic">Agl<sup>−/−</sup></span> compared to WT mice in both the light condition (<b>A2</b>) and overall (<b>A4</b>), and that GHF-201 corrects this TEE reduction in <span class="html-italic">Agl</span><sup>−/−</sup> mice in the light. ((<b>B</b>)—Activity) Ambulatory activity (<b>B1</b>) and wheel running (<b>B2</b>) were also increased (corrected), or not affected, by GHF-201 as indicated. ((<b>C</b>)—Food and Water Intake) Food intake (<b>C1</b>) was not significantly affected, and water intake ((<b>C2</b>), total cumulative values are shown in the bar graphs) was increased, mainly in the dark period, by GHF-201. Statistical significance of mean differences was determined by One Way ANOVA with repeated measures with a Tukey post hoc test. *, 0.01 <span class="html-italic">< p</span> < 0.05; **, 0.001 < <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001; ****, <span class="html-italic">p</span> < 0.0001; ns, non-significant.</p> "> Figure 5
<p>(<b>A</b>) Representative transmission electron microscopy images of longitudinal sections of <span class="html-italic">gastrocnemius</span> muscle collected from 8-month-old animals treated as indicated. Note granular glycogen material interspersing sarcomeres in the section from untreated <span class="html-italic">Agl<sup>−/−</sup></span> mouse. Also note variable width of sarcomeres in <span class="html-italic">Agl<sup>−/−</sup></span> mouse sample. These ultrastructural phenotypes were partially corrected in GHF-201-treated <span class="html-italic">Agl<sup>−/−</sup></span> mouse. (<b>B</b>) Quantification of area percent of glycogen based on analysis of TEM images from n = 3 animals. ****, significant differences between arms (<span class="html-italic">p</span> < 0.0001, Two Way ANOVA (factors are “animal” and “arm”) with Tuckey post hoc correction). (<b>C</b>) Biochemical quantification of <span class="html-italic">Gastrocnemius</span> glycogen. Glycogen levels in muscle tissues collected from n = 3 mice/arm were determined by Merck’s glycogen assay kit and statistically analyzed by One Way ANOVA with Tuckey post hoc correction. *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01, ns, <span class="html-italic">p</span> < 0.08.</p> "> Figure 6
<p>Skin fibroblasts from 3 GSD3 and 3 HC individuals were cultured in 96-well plates for 48 h in starvation medium without FBS and glucose. Subsequently, cells were live-stained with (<b>A</b>,<b>B</b>) PAS reagent to quantify intracellular glycogen, or (<b>C</b>–<b>E</b>) a mix of fluorescent dyes which included Hoechst, Calcein-AM, TMRE, and Lysortracker red to, respectively, stain nuclei (blue), cytoplasm (green), respiring mitochondria (yellow), and lysosomes (red). Multiple images from the live-stained cells were automatically obtained by an Operetta G1 image analyzer under environmentally controlled conditions. Shown are representative images and their quantification in relative units. Statistical significance of differences was determined by Two Way ANOVA with Tuckey post hoc correction. *, <span class="html-italic">p</span> < 0.05; **, 0.001 < <span class="html-italic">p</span>< 0.01; ***, <span class="html-italic">p</span> < 0.001; ns, not significant.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. In Vivo Studies
2.2. Ultrastructural Electron Microscopy Studies
2.3. Biochemical Glycogen Quantification
2.4. High Content Analysis of GSDIII Patient Fibroblasts
2.5. Statistical Analysis
3. Results
3.1. Animal Weight
3.2. Locomotor Studies
3.3. Blood Metabolic Panel
3.4. In Vivo Metabolic Profile
3.5. Muscle Glycogen Quantification
3.6. Glycogen Levels, Lysosomal and Mitochondrial Features of GSDIII Patient-Derived Skin Fibroblasts
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mishra, K.; Sweetat, S.; Baraghithy, S.; Sprecher, U.; Marisat, M.; Bastu, S.; Glickstein, H.; Tam, J.; Rosenmann, H.; Weil, M.; et al. The Autophagic Activator GHF-201 Can Alleviate Pathology in a Mouse Model and in Patient Fibroblasts of Type III Glycogenosis. Biomolecules 2024, 14, 893. https://doi.org/10.3390/biom14080893
Mishra K, Sweetat S, Baraghithy S, Sprecher U, Marisat M, Bastu S, Glickstein H, Tam J, Rosenmann H, Weil M, et al. The Autophagic Activator GHF-201 Can Alleviate Pathology in a Mouse Model and in Patient Fibroblasts of Type III Glycogenosis. Biomolecules. 2024; 14(8):893. https://doi.org/10.3390/biom14080893
Chicago/Turabian StyleMishra, Kumudesh, Sahar Sweetat, Saja Baraghithy, Uri Sprecher, Monzer Marisat, Sultan Bastu, Hava Glickstein, Joseph Tam, Hanna Rosenmann, Miguel Weil, and et al. 2024. "The Autophagic Activator GHF-201 Can Alleviate Pathology in a Mouse Model and in Patient Fibroblasts of Type III Glycogenosis" Biomolecules 14, no. 8: 893. https://doi.org/10.3390/biom14080893