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. 2020 Jun 10:18:199-214.
doi: 10.1016/j.omtm.2020.05.026. eCollection 2020 Sep 11.

Enzyme Replacement Therapy Can Reverse Pathogenic Cascade in Pompe Disease

Naresh Kumar Meena  1 , Evelyn Ralston  2 , Nina Raben  1 , Rosa Puertollano  1
Affiliations

Enzyme Replacement Therapy Can Reverse Pathogenic Cascade in Pompe Disease

Naresh Kumar Meena et al. Mol Ther Methods Clin Dev. .

Abstract

Pompe disease, a deficiency of glycogen-degrading lysosomal acid alpha-glucosidase (GAA), is a disabling multisystemic illness that invariably affects skeletal muscle in all patients. The patients still carry a heavy burden of the disease, despite the currently available enzyme replacement therapy. We have previously shown that progressive entrapment of glycogen in the lysosome in muscle sets in motion a whole series of "extra-lysosomal" events including defective autophagy and disruption of a variety of signaling pathways. Here, we report that metabolic abnormalities and energy deficit also contribute to the complexity of the pathogenic cascade. A decrease in the metabolites of the glycolytic pathway and a shift to lipids as the energy source are observed in the diseased muscle. We now demonstrate in a pre-clinical study that a recently developed replacement enzyme (recombinant human GAA; AT-GAA; Amicus Therapeutics) with much improved lysosome-targeting properties reversed or significantly improved all aspects of the disease pathogenesis, an outcome not observed with the current standard of care. The therapy was initiated in GAA-deficient mice with fully developed muscle pathology but without obvious clinical symptoms; this point deserves consideration.

Keywords: Pompe disease; acid alpha glucosidase; autophagy; enzyme replacement therapy; glycogen; lysosomal targeting; mTORC1/AMPK signaling; metabolome; muscle.

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Figures

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Graphical abstract
Figure 1
Figure 1
AT-GAA Cleared Glycogen Accumulation and Restored Normal Levels of Endosomal/Lysosomal Membrane Markers in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Glycogen content in muscles from WT, untreated KO (KO), and treated KO (KO-ERT) mice (n = 6 for each group). (B) Muscle strength was assessed using grip-strength test after 8 and 9 administrations of the compound (n = 5 WT; n = 4 KO; n = 7 KO-ERT). (C) GAA activity in muscle tissues. (D) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice (n = 6 for each group) with the indicated antibodies. GAPDH was used as loading control. Each data point represents an individual mouse. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 2
Figure 2
AT-GAA Reversed the Levels of the Autophagic Markers and Alleviated the Burden of Accumulated Protein Aggregates and ER-Stress in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group; n = 5 for western blot with LC3 antibody). (B) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with K63-linked ubiquitin specific antibody (n = 4 for each group). Western blot with anti-GAPDH and Ponceau S staining were used as loading controls. (C) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with anti-Grp 78 antibody (ER-stress marker; n = 4 for each group); GAPDH was used as loading control. Each data point represents an individual mouse. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 3
Figure 3
AT-GAA Eliminated Autophagic Buildup in Vast Majority of Myofibers from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. Confocal microscopy images of single muscle fibers immunostained with lysosomal marker LAMP1 (red) and autophagosomal marker LC3 (green); nuclei are stained with Hoechst dye (blue). (A) Abundant dot-like LAMP1-positive structures (red) are seen in a fiber from a WT mouse; LC3-positive autophagosomes are barely detectable in control fibers (n = 60 from 3 mice). (B and C) Enlarged lysosomes (red) and autophagic buildup (the multicolored areas in the core of muscle fibers) are detected in virtually all myofibers from KO mice; the images show two neighboring fibers with autophagic buildup located in different focal planes; therefore, the buildup is seen in the left fiber in (B), and in the right fiber in (C) (n = 85 from 4 untreated KO mice). See also Video S1. (D–G) The 4 right panels show the representative images of myofibers from treated KO mice (n = 217 from 4 mice). Myofibers shown in (D), as well as the left fiber in (G), are considered normal; near normal fibers are shown in (E) and (F) (n = 182; ∼84% of normal or near normal fibers). The right fiber in (G) contains a more typical buildup (n = 35; ∼16%). The dot plot indicates the percentage of normal/near normal fibers from treated KO compared to untreated KO mice. Graphs represent mean ± SD. Scale bars, 20 μm. (See also Figure S1).
Figure 4
Figure 4
AT-GAA Improved Muscle Quality and Architecture in KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. The samples were analyzed by second harmonic generation (SHG) and two-photon-excited fluorescence (2PEF) imaging. SHG reveals the position and organization of myosin heavy chain, while (2PEF) reveals mitochondria and autofluorescent particles. Every fiber from untreated KO mice (n = 3) show long longitudinal interruptions of the SHG image filled with 2PEF-positive particles (arrows); these areas correspond to the space occupied by the autophagic buildup. In contrast, most fibers from the treated KO mice (n = 3) do not show this defect; nevertheless, small interruptions of the SHG images can be seen in some fibers (arrows). Note that most fibers from treated mice have a larger diameter compared to that in untreated KO. Scale bars, 25 μm.
Figure 5
Figure 5
AT-GAA Reversed the Level of Galectin 3, a Marker of Lysosomal Damage, in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group). Only galectin 3 was increased in muscle from untreated KO mice; the level of galectin 3 was reduced on therapy and reached the WT control value. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (B) Western blot of lysates from the diaphragm (top) and heart (bottom) of WT and untreated KO (KO) mice with anti-galectin 3 antibody. (C) Western blot of muscle lysates from untreated KO and muscle-specific autophagy-deficient KO mice (DKO) with anti-galectin 3 antibody. Efficient suppression of autophagy in skeletal muscle of DKO mice is indicated by the absence of LC3-II band. The blots are composite images; the samples were run on the same gel. Source data are available online for this figure. GAPDH was used as loading control. (D) Quantification of galectin 3 in serum from the WT and KO mice by ELISA. Student’s t test was used for statistical analysis. Data are mean ± SD. ∗p < 0.05 (n = 6).
Figure 6
Figure 6
AT-GAA Improved AMPK-TSC2 Signaling in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group for LKB1; n = 6 for each group for p-AMPKα T172). (B and C) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group). GAPDH was used as loading control. Each data point represents an individual mouse. (D) A diagram showing the position of the analyzed proteins. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 7
Figure 7
AT-GAA Had a Modest Effect on mTORC1 Signaling in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with anti-AKT and anti-PRAS40 antibodies (n = 4–6 for each group). (B) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4–6 for each group). Different forms of 4EBP1, phosphorylated (p-4EBP1T37/46 and p-4EBP1S65), non-phosphorylated (non-p-4EBP1T46), and total were analyzed. No changes in the mTOR activity are detected in treated compared to untreated KO samples; both exhibit diminished mTOR activity when compared to WT as shown by the decrease in the levels of phosphorylated S6 and 4EBP1, downstream targets of mTORC1 (top panels). A decrease in both non-p-4EBP1T46 and total is seen in treated compared to untreated KO muscle (lower panels). GAPDH was used as loading control. Each data point represents an individual mouse. (C) A diagram showing the position of the analyzed proteins. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 8
Figure 8
AT-GAA Had a Modest Effect on Muscle Proteostasis in KO Mice KO mice received 12 bi-weekly i.v. administrations of AT-GAA. Age and sex-matched WT and untreated KO mice were used for the comparisons. Muscle biopsies were collected 7–10 days after the last administration. (A) Surface sensing of translation (SUnSET) analysis was used to evaluate the rate of protein synthesis. The animals were injected intraperitoneally (i.p.) with puromycin (the aminoacyl-tRNA analog) 30 min prior to sacrifice. Western blot of muscle lysates with anti-puromycin antibody was then used to detect the incorporation of puromycin into nascent polypeptides. Total intensity of puromycin-labeled polypeptides was quantified. The increased protein translation is observed in both untreated and treated KO samples. Western blot with anti-GAPDH and Ponceau S staining were used as loading controls. (B) Western blot of muscle lysates from WT, untreated-, and treated KO muscle shows a decrease in the p-eIF2α S51/eIF2α ratio (consistent with the increase in protein synthesis) in both untreated and treated KO samples. (C) Western blot of total lysates from WT, untreated-, and treated KO muscle shows increased levels of proteasome 26S subunit, ATPase 1 (PSMC1), and alpha 5 (PSMA5) subunits in the KO; the level of PSMC1 returned to normal following ERT, whereas the level of PSMA5 did not (n = 5 for each condition. (D) The proteasome activity was measured in proteasome-enriched fractions isolated from WT, untreated-, and treated KO muscle extracts. The activity was significantly improved following ERT but still remained elevated compared to the WT controls. The results are shown in relative fluorescence units (RFU)/mg protein (n = 3 for each group). Note that the lysates for (B) and (C) were the same as those used in Figures 1, 2, 5,6, and 7 (n = 4–5 for each group). Data are mean ± SD. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 9
Figure 9
Capillary Electrophoresis-Mass Spectrometry Analysis of Skeletal Muscle from WT, Untreated-, and Treated KO Mice (A) Principal-component analysis (PCA) of the metabolomic datasets of the skeletal muscle of WT, untreated-, and treated KO mice (n = 6 for each of the three groups). Plots of the three groups are clearly separated on the x axis. (B) A heatmap of hierarchical cluster analysis of the metabolite changes of the skeletal muscle of WT, untreated-, and treated KO mice. The heatmap patterns between WT (6 right lanes) and KO (6 middle three lanes) are clearly distinguishable. The heatmap pattern of treated KO samples (6 left lanes) appears to be closer to that of the WT. Red color indicates relatively high content of metabolites; green indicates relatively low content of metabolites.
Figure 10
Figure 10
Metabolite Changes in Skeletal Muscle from WT, Untreated-, and Treated KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A–D) The levels of the metabolites involved in the glycolytic pathway (A), TCA cycle (B), carnitine, ATP, and total amino acids (C), and the levels of glycogen precursors (D) in the three groups. (E) Western blot analysis of whole muscle lysates from WT, untreated-, and treated KO mice with indicated antibodies. An increased phosphorylation (inactivation) of glycogen synthase (p-GS S641) in KO muscle (consistent with our previously reported data10) is reversed in treated KO mice (n = 4 for each group). Each lane represents a sample from a single mouse. Data are mean ± SD. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.
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