Mechanism of neuromuscular dysfunction in Krabbe disease

Abstract

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher-Thy1.1-yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Keywords: Akt; Krabbe disease; neuromuscular junction; neuropathy; proteosome; psychosine.

Figure 1.

Muscle atrophy in the twitcher mouse. A, B, D, E, Representative pictures of the right leg of P30 twitcher (Twi, A, B) and wild-type (WT, D, E) mice. A reduction in mass is visible in twitcher muscles (arrows). C, The weight of the twitcher gastrocnemius muscle was quantitated at P7, P15, P30, and P45. n = 4–6 mice per genotype per time point. *p < 0.05, ANOVA.

Figure 2.

Decreased isometric force, fibrosis, and decreased fiber size in twitcher mice. A, Measurement of isometric twitching force was done on TA muscles of twitcher (Twi) and wild-type (WT) mice at P15 and P30. Force (in grams) was normalized against the mass of the muscle (in milligrams). n = 3 muscles per genotype per time point. *p < 0.01, ANOVA. C, E, Fibrosis was histologically visible after Gomori's trichrome staining of coronal sections of gastrocnemius muscles. B, D, F, Hematoxylin–eosin staining served to quantify reductions of fiber cross-sectional area (asterisks in B) in serial coronal sections of gastrocnemius muscles. n = 3 muscles per genotype per time point.

Figure 3.

Absence of denervation in twitcher muscles. A–K, NMJs were detected by confocal imaging using soleus muscle preparations from the twitcher–YFPax and wild-type (WT) YFPax reporter mouse models at P7 (A, B), P15 (C, D), and P30 (F–K) after staining postsynaptic neuromuscular membranes with Alexa Fluor-555 BTX (in red). Arrowhead points to a denervated NMJ in twitcher (TWI) muscle. L–R, Endplates were studied in whole-mount preparations of mutant and wild-type diaphragm muscles. Axons were labeled using anti-NF-M antibodies (in red) and Alexa Fluor-488 BTX (in green) and analyzed by confocal imaging and orthogonal reconstructions (planes x–z, y–z). E, O, The proportion (as percentage of the total) of complete denervated (BTX⁻/YFP⁺) and innervated (BTX⁺/YFP⁺) endplates in P30 soleus (E) and P35 diaphragm (O) muscles was calculated and found not significantly different in twitcher mice (n = 30–50 NMJs per genotype).

Figure 4.

Axonal terminals and NMJs express high levels of activated caspase-3 in the twitcher soleus muscle. A–J, Levels of activated caspase-3 (a-Casp.3, in red) were determined by immunohistochemistry of NMJs after confocal imaging using soleus muscle preparations from the twitcher–YFPax and wild-type (WT) YFPax reporter mouse models at P7 (A, B), P15 (C, D), and P30 (E–J). Arrowhead points to NMJs with activated caspase-3 in twitcher (TWI) muscle. K, The proportion (as percentage of the total) of NMJs with detectable levels of active caspase-3 was calculated in muscle preparations at P7, P15, and P30. n = 3 muscles per genotype per time point. *p < 0.01, ANOVA.

Figure 5.

Soleus NMJs are atrophied in twitcher mice and re-express the AChR-γ subunit. A–D, The morphology of NMJs was determined by 3D confocal reconstruction of soleus muscle preparations from the twitcher–YFPax and wild-type YFPax reporter mouse model at P30. Images in A show two stereotypical reconstructed images for a twitcher (TWI) and wild-type (WT) NMJ. Images are at the same magnification. Arrows point to the axonal terminals of the junctions. The frequency of smaller NMJ (as percentage of the total, B), the exposed surface area (in square micrometers, C), and total volume (in cubic micrometers, D) were calculated. n = 3 muscles per genotype. *p < 0.05, t test). E, The expression of the β and γ AChR subunit mRNA was analyzed by real-time PCR in RNA extracts from twitcher (TWI) and wild-type (WT) gastrocnemius muscles at P7, P15, and P30. Data are expressed as fold changes in twitcher over wild type.

Figure 6.

Extensor digitorum longus motor units exhibit synaptic failure in twitcher mice. MEPPs were recorded continuously on preparations of extensor digitorum longus isolated from P15 (A–H) and P30 (I–P) twitcher (Twi) and wild-type (WT) mice (n = 6 mice per genotype). Amplitude (in millivolts, B, J), frequency (in events per second, D, L), and rise (F, N) and decay (H, P) constants (in milliseconds) were calculated at each time point. Distribution histograms are shown in A, C, E, G, I, K, M, and O, indicating the number of muscle cells studied for each parameter. *p < 0.01, t test.

Figure 7.

Defective Akt signaling drives muscle atrophy in twitcher mice. A–E, Protein extracts were prepared from gastrocnemius muscles from twitcher (TWI) and wild-type (WT) mice at P7, P15, and P30. Extracts were immunoblotted for phosphorylated (Ser473; A) and total (B) Akt, phosphorylated and total 4EBP1 (D), and phosphorylated (Ser9) and total (E) GSK3β. Real-time PCR measured the abundance of mRNA for the MAFbx gene (C). n = 3 muscles per genotype per time point. *p < 0.01, ANOVA. F–H, Psychosine was measured by tandem mass spectrometry in differentiated cultures of myotubes (F) and total lipid extracts from gastrocnemius, gluteus, and diaphragm muscles in twitcher (G) and GLD (H) animals. Age-matched controls were used for each determination. n = 3–4 muscles (or culture extracts) per genotype per time point. *p < 0.01, ANOVA and t test. I, Cultures of wild-type myotubes were incubated with 5 μm psychosine (PSY) or with a mock vehicle (0.005% ethanol/PBS) solution before measuring the level of phosphorylation of Akt by immunoblotting. n = 3 samples per experimental condition point. *p < 0.05, t test. J, The illustration proposes a mechanistic model of muscle atrophy in Krabbe disease, including the key elements described by the analyses of the twitcher mouse. Psychosine is likely acting at multiple levels, by blocking fast axonal transport (FAT) in motor axons, affecting myelin and Schwann cells in peripheral nerves, and repressing Akt activation in muscle cells. Deficient electrical conduction at the endplate likely drives the re-expression of the embryonic AChR-γ subunit at the postsynaptic membrane, contributing to inefficient neuromuscular activity. Reduced endplate activity in conjunction with in situ accumulation of psychosine reduces Akt activity further, driving twitcher muscles toward atrophy via proteosome degradation and reducing muscle hypertrophy via protein synthesis shutdown. p, Phosphorylated; t, total.