Peripheral nerve and neuromuscular junction pathology in Pompe disease

Abstract

Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of Pompe disease.

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Figures

Figure 1.

Widespread disruption of diaphragmatic NMJs in Pompe animals. Confocal images of AChR distribution in the diaphragm of representative (A and C) 9-month-old old WT 129SVE and (B and D) age-matched Gaa−/− mice. Lower magnification images from the same tissue samples show triple immunolabeling with anti-NFH (magenta), anti-synaptotagmin (green) and alpha-bungarotoxin (red). The white arrow in (C) indicates colocalization of presynaptic synaptotagmin and α-bungarotoxin labeling. (D) Representative area of NMJs in Gaa−/− diaphragm show marked reduction in colocalization of pre- and postsynaptic labels. White arrows indicate loss of presynaptic staining in motor endplates. Scale bars represent 10 μm.

Figure 2.

Synaptic pathology in the diaphragm of Pompe animals. (A) Mean diaphragm endplate size is significantly larger in Gaa−/− (gray column) mice compared with age-matched WT controls (white column; ****P ≤ 0.0001, Mann–Whitney t-test, two-tailed; n = 4 biological replicates per group). (B) Histogram of endplate size showing frequency distribution of endplate area across the diaphragm of WT and Gaa−/−animals (n = 4). (C) The mean percentage of fragmented diaphragm endplates in Gaa−/− (gray) and WT (white) mice (*P ≤ 0.05, Mann–Whitney t-test, two-tailed). (D) Quantification of multi- (gray) or single- (white) innervated endplates within the diaphragm of Gaa−/− compared with age-matched control mice (two-way repeated measures ANOVA, n = 4 biological replicates per group). (E) Quantification of NMJ innervation in the diaphragm of 9-month-old Gaa−/− mice relative to age-matched controls (two-way ANOVA with Sidak's multiple comparison test, n = 4 animals per group).

Figure 3.

Alterations of nerve morphology in the phrenic nerve of Pompe mice. Representative cross section of the phrenic nerve from (A) WT mice and (B) Gaa−/− mice. (C, D) Significantly lower average G-ratio in phrenic nerves from 9-month-old Gaa−/− mice (0.4490 ± 0.0080, n = 447 fibers from four independent animals) compared with age-matched WT control mice (mean ratio 0.6961 ± 0.0037, n = 300 fibers from three independent animals), indicating thicker myelin sheaths relative to the axon diameter (C; ****P ≤ 0.0001; two-tailed, unpaired t-test), with lower G-ratios across a range of axon calibers (D). Scale bars represent 50 μm.

Figure 4.

Widespread NMJ disruption in the TA of 9-month-old Pompe animals. Confocal micrographs of individual postsynaptic sites in in the TA of (A, C) 9-month-old WT and (B, D) 9-month-old Gaa−/− mice, as visualized by the binding of α-bungarotoxin. Lower magnification images of the distribution of NFH (magenta), synaptotagmin (green) and alpha-bungarotoxin (red). The white arrow in (C) indicates colocalization of presynaptic synaptotagmin and α-bungarotoxin labeling in AChR clusters. (D) Representative area of NMJs in Gaa−/− diaphragm show marked reduction in colocalization of pre- and postsynaptic labels. Scale bars represent 10 μm.

Figure 5.

Synaptic pathology in hindlimb muscle of 9-month-old Pompe animals. (A) The mean TA endplate size is significantly larger in Gaa−/− (gray column) mice compared with age-matched WT controls (white column; P ≤ 0.05, t-test, two-tailed; n = 4 biological replicates per group). (B) Histogram of endplate size showing frequency distribution of endplate area across the TA of WT and Gaa−/− animals (n = 4). (C) The mean percentage of all measured TA endplates that were fragmented in Gaa−/− (gray) and WT (white) mice (P ≤ 0.05, Mann–Whitney t-test, two-tailed). (D) Quantification of multi- (gray) or single- (white) innervated endplates within the diaphragm of Gaa−/− compared with age-matched control mice (two-way repeated measures ANOVA, n = 4 biological replicates per group). (E) Quantification of NMJ innervation in the TA of 9-month-old Gaa−/− mice relative to age-matched controls (two-way ANOVA with Sidak's multiple comparison test, n = 4 animals per group).

Figure 6.

Alterations of tissue morphology in the sciatic nerve of Pompe mice. Morphological analysis of the sciatic nerve from (A) WT and (B) Pompe (Gaa−/−) mice highlights altered nerve fiber distribution and increased extracellular space between individual nerve fibers (asterisks). (C,D) Significantly lower average G-ratio in sciatic nerves from 9-month-old Gaa−/− mice (0.4111 ± 0.0055, n = 850 fibers from four independent animals) compared with age-matched WT control mice (mean ratio 0.5431 ± 0.0048, n = 869 fibers from three independent animals), indicating thicker myelin sheaths relative to the axon diameter (C; ****P ≤ 0.0001; two-tailed, unpaired t-test), with lower G-ratios across a range of axon calibers (D). Scale bars represent 50 μm.

Figure 7.

Loss of Gaa impairs neuromuscular function in 9-month-old mice. In situ analysis of muscle torque production revealed a significant decrease in performance of Gaa−/− mice relative to age-matched WT mice across all frequencies tested (*P ≤ 0.05).

Figure 8.

Accumulation of LAMP1 in Schwann cells in the sciatic nerve of affected mice. Sciatic nerves from WT (A, C and E) and Gaa−/− (B, D and F) mice were doubled, labeled with anti-S100 (red) and anti-LAMP1 (green) antibodies. Nuclei were stained with DAPI (blue). Arrows indicate the perinuclear area of S100-positive Schwann cells, while arrowheads indicate the paranodal region of myelin internodes. The majority of LAMP1 in Gaa−/− is detected as perinuclear or paranodal puncta in S100-positive Schwann cells, with pronounced abnormal accumulation in a subset of the Schwann cells. Paranodal LAMP1 vesicles in Gaa−/− also appear less punctate. Scale bars = 10 μm. Blue = DAPI, green = S100, red =LAMP1.

Figure 9.

Neuropathic changes in the sciatic nerve of Pompe animals. Representative western blot images and quantification normalized to α-tubulin for select proteins within the sciatic nerve of 9-month-old WT and Gaa−/− mice. (A) LAMP1 (P ≥ 0.05; two-tailed, unpaired t-test). (B) LAMP2 (P ≤ 0.05; two-tailed, unpaired t-test). (C) MBP (P ≥ 0.05; two-tailed, unpaired t-test). (D) Growth-associated protein 43 kDa (P ≥ 0.05; two-tailed, unpaired t-test). (E) Non-phosphorylated neurofilament heavy and medium polypeptide (P ≤ 0.05; two-tailed, unpaired t-test).