The value of muscle biopsies in Pompe disease: identifying lipofuscin inclusions in juvenile- and adult-onset patients

Erin J Feeney, Stephanie Austin, Yin-Hsiu Chien, Hanna Mandel, Benedikt Schoser, Sean Prater, Wuh-Liang Hwu, Evelyn Ralston, Priya S Kishnani, Nina Raben, Erin J Feeney, Stephanie Austin, Yin-Hsiu Chien, Hanna Mandel, Benedikt Schoser, Sean Prater, Wuh-Liang Hwu, Evelyn Ralston, Priya S Kishnani, Nina Raben

Abstract

Background: Pompe disease, an inherited deficiency of lysosomal acid alpha-glucosidase (GAA), is a metabolic myopathy with heterogeneous clinical presentations. Late-onset Pompe disease (LOPD) is a debilitating progressive muscle disorder that can occur anytime from early childhood to late adulthood. Enzyme replacement therapy (ERT) with recombinant human GAA is currently available for Pompe patients. Although ERT shows some benefits, the reversal of skeletal muscle pathology - lysosomal glycogen accumulation and autophagic buildup - remains a challenge. In this study, we examined the clinical status and muscle pathology of 22 LOPD patients and one atypical infantile patient on ERT to understand the reasons for muscle resistance to ERT.

Results: The patients were divided into three groups for analysis, based on the age of onset and diagnosis: adult-onset patients, juvenile-onset patients, and those identified through newborn screening (NBS). The areas of autophagic buildup found in patients' biopsies of all three groups, contained large autofluorescent inclusions which we show are made of lipofuscin, an indigestible intralysosomal material typically associated with ageing. These inclusions, analysed by staining, spectral analysis, time-resolved Fluorescence Lifetime Imaging (FLIM), and Second Harmonic Generation (SHG) imaging, were the major pathology remaining in many fibers after ERT. The best outcome of ERT both clinically and morphologically was observed in the NBS patients.

Conclusions: The muscle biopsy, in spite of its shortcomings, allowed us to recognize an underreported, ERT-resistant pathology in LOPD; numerous lysosomes and autolysosomes loaded with lipofuscin appear to be a hallmark of LOPD skeletal muscle. Lipofuscin accumulation - a result of inefficient lysosomal degradation - may in turn exacerbate both lysosomal and autophagic abnormalities.

Figures

Figure 1
Figure 1
Montage of confocal fluorescence images of unstained fibers from patient NBSL9a, showing numerous autofluorescent inclusions, single and in clusters, in the core of two fibers. Bar: 50 μm.
Figure 2
Figure 2
Autofluorescent lipofusin inclusions in muscle biopsies from LOPD. (a) LOPD fiber (pt. NBSL9a) viewed in fluorescence (top) and transmitted light (bottom) shows autofluorescent inclusions directly surrounded by myofibrils. Fluorescence was excited at 488 nm and collected from 467 to 499 nm. Transmitted light is with DIC contrast. Bar: 25 μm. (b) LOPD fiber (pt. NBSL2) with prominent inclusions. The fiber was stained with LAMP2 (lysosomes: green) and LC3 (autophagosomes: red). Some inclusions are seen within the lysosome or autolysosome (arrows) whereas others appear free in the cytoplasm (asterisks). Bar: 10 μm. (c) Autofluorescent inclusions stain positive for Oil Red. The fiber (isolated from muscle biopsy of pt. D3) was also stained with LAMP2 (green). Bar: 10 μm. (d) Sudan Black B staining demonstrates lipofuscin accumulation in a fiber from pt. D3. Bar: 10 μm.
Figure 3
Figure 3
Analysis of inclusions in muscle biopsies from an LOPD patient (NBSL9a) and a GAA-KO mouse. (a) Confocal images of a muscle fiber from a LOPD biopsy with excitation at 405, 488, and 568 nm respectively. The last panel shows the sum of the three images. Autofluorescent particles are excited by each of the wavelengths while a Hoechst-stained nucleus (asterisk) is only excited at 405 nm. An arrowhead points to a small normal-looking lysosome at a pole of the nucleus while an arrow points to the end of the particle row with a small brighter area. (b) Two-photon excited fluorescence of LOPD and GAA-KO fibers recorded in spectral mode on a confocal microscope. Fluorescence emission spectra from 460 to 660 nm were displayed for the areas within colored boxes and plotted in Excel. There are minor differences between the human and mouse samples - LOPD fibers have particles that stand out in brightness and are slightly red-shifted (purple box and spectrum); these particles are commonly found at the end of the row of inclusions (see also arrows in panel a). Background autofluorescence corresponds to mitochondria in I bands [30]. (c) FLIM analysis confirms the heterogeneity of autofluorescent particles in both GAA-KO and LOPD fibers. Left panels show the intensity of fluorescence emission while right panels are pseudo-colored to represent average lifetimes. The bright particles in the LOPD fiber (arrows) are similar to those in the purple box shown in b; their average lifetime is shorter (blue color). The wide spectra (a & b) support the notion that the inclusions consist of lipofuscin; FLIM analysis suggests that the particles may mature as the disease progresses. Bars: 10 μm (a); 50 μm (b).
Figure 4
Figure 4
Second Harmonic Generation (SHG) microscopy of a single fiber from an LOPD muscle biopsy. Stacks of SHG and autofluorescence images were recorded with a step of 0.85 μm to image the whole fiber from top to bottom. SHG (green) shows myosin bands, whereas autofluorescence (red) shows the inclusions. Several images from one series are shown here. The distance from the top of the fiber is indicated. As the focal plane encounters the first inclusions, the interruption of the myosin bands is clear. The interruption becomes the “black hole” of autophagic areas (see text). As the focal plane reaches the other side of the particles, thin partial myosin bands are seen. Arrowheads point to the interruptions, total or partial of the myosin bands; arrows highlight defects in the myofibril alignment. Bar: 25 μm.
Figure 5
Figure 5
Autophagic abnormalities and autofluorescent inclusions in adult-onset patients. a-c: Muscle fibers were stained for lysosomal marker LAMP2 (green) and autophagosomal marker LC3 (red). (a) LOPD fiber (pt. D16) shows mild lysosomal enlargement and clusters of autophagosomes. (b) Autophagic buildup and autofluorescent inclusions represent a major pathology in this fiber (pt. D7). (c) Autofluorescent inclusions are seen within the LAMP2-positive structures (pt. D7). Bar in a-c: 10 μm. (d) Both autophagic buildup and inclusions are missed by routine histology; the image shows epon-embedded PAS-stained section of muscle biopsy from pt. D15 (10x).
Figure 6
Figure 6
Analysis of muscle biopsies from an adult-onset patient D9. (a) H&E stained section of the first biopsy (taken 6 years prior to initiation of ERT) shows vacuolation in ~20-25% fibers (10x). The second biopsy was performed after 6 years of ERT (b-f). (b) H&E staining shows mostly vacuolated fibers (10x) (note, some of the large “holes” are likely freeze artefacts). (c) EM demonstrates the presence of autophagic buildup and “pale” areas (arrows) in the surrounding relatively well preserved fibers (transverse section). Bar: 2 μm. (d-f) Muscle fibers were stained for lysosomal marker LAMP2 (green) and autophagosomal marker LC3 (red). Nuclei are stained with Hoechst (blue). LAMP2/LC3 staining demonstrates prominent autophagic accumulation with inclusions in most fibers; these abnormalities are commonly seen in fibers with mild (d) or no (e and f) lysosomal enlargement. Prominent lysosomal enlargement is seen in occasional fibers (e; arrowheads). Bar: 10 μm.
Figure 7
Figure 7
Analysis of muscle biopsies from juvenile-onset patients. Muscle fibers were stained for lysosomal marker LAMP2 (green) and autophagosomal marker LC3 (red). LAMP2/LC3 immunostaining demonstrates variability of muscle fiber involvement: completely destroyed fibers in pts. HM3 (a) and HM1 (d); a typical well- preserved fiber in pt. CLINM (b); fibers with inclusions in pts. HM1 and HM5 (c and e respectively), and a fiber with lysosomal enlargement in pt. HM1 (c; arrowheads). Bar: 10 μm.
Figure 8
Figure 8
Analysis of muscle biopsy from a patient (D3) with atypical infantile-onset Pompe disease. Muscle fibers were stained for lysosomal marker LAMP2 (green) and autophagosomal marker LC3 (red). LAMP2/LC3 immunostaining demonstrates variability of muscle fiber involvement: fibers with autophagic accumulation (for example, top fiber in panel a) and inclusions (b), in addition to largely intact muscle fibers (c) interspersed with completely destroyed fibers (a and c). Bar: 10 μm.

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