Nucleolar characteristics of reducing bodies in reducing body myopathy

Abstract

Reducing body myopathy is a rare muscle disease defined by abnormal inclusions in affected muscle fibers that can be stained with menadione-nitroblue tetrazolium. The origin of these inclusions has not been determined. Here we show that reducing bodies bear characteristics of nucleoli. Ultrastructurally, muscle biopsy specimens of a patient with adult-onset reducing body myopathy showed granular structures of reducing bodies with features similar to the granules of nucleoli, which consisted of pre-ribosomes. In addition, reducing bodies were positive for histochemistry of argyrophilic nucleolar organizer regions (a method for detecting the areas where ribosomal RNA is actively transcribed in the nucleolus), and for antibodies against nucleoli and nuclear ribonucleoprotein. The current findings suggest that reducing bodies contain pre-ribosomes and their associated proteins of the nucleolus and that formation of reducing bodies may result from defects of processing and assembly of ribosomes.

Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy

“…Immunohistochemistry using antibodies directed against FHL1 confirmed that the intracytoplasmic inclusion bodies were highly enriched for FHL1 immunoreactivity ( Figure 2B and Supplemental Figure 2D), whereas it was distributed without focal accumulations in normal muscle sections ( Figure 2A and Supplemental Figure 2C). Under higher magnification, the FHL1-positive inclusions were seen throughout myofibers but were often also localized in close association with the myonuclei (Figure 2C), consistent with their appearance on electron microscopy ( Figure 2D and Supplemental Figure 2A) and with previous histopathological observations in this condition (7,(13)(14)(15)(16). We also confirmed that the intracytoplasmic inclusions labeled positively for other proteins identified in the mass spectrometry analysis, including desmin and ubiquitin ( Figure 2, E and F) (2,7,13,16,18).…”

“…Under higher magnification, the FHL1-positive inclusions were seen throughout myofibers but were often also localized in close association with the myonuclei (Figure 2C), consistent with their appearance on electron microscopy ( Figure 2D and Supplemental Figure 2A) and with previous histopathological observations in this condition (7,(13)(14)(15)(16). We also confirmed that the intracytoplasmic inclusions labeled positively for other proteins identified in the mass spectrometry analysis, including desmin and ubiquitin ( Figure 2, E and F) (2,7,13,16,18). Further analysis of the localization of FHL1 immunoreactivity within skeletal muscle from the RBM patients showed FHL1 to be localized to the I-band/Z disk in fibers without obvious inclusions, indistinguishable from the localization in normal muscle ( Figure 2G).…”

“…In addition, the aggregates trap the interacting protein MyBP-C as well as other proteins that do not normally interact, such as dystrophin and LAMP1 ( Figure 5F, Supplemental Figure 2E, and Supplemental Figure 3F). These proteins have previously been demonstrated to be part of the inclusions in patient biopsies by immunohistochemical analysis (2,7,13,16,18). Our transfection experiments in C2C12 cells demonstrate that the presence of the mutant FHL1 initiates this process, leading to the incorporation of these other bystander proteins into the inclusions.…”

“…Notably, the inclusions labeled prominently for the intermediate filament desmin, ubiquitin, the luminal endoplasmic reticulum chaperone GRP78, as well as for γ-tubulin, all of which are consistent with aggresome formation (18). Although a large number of diverse proteins have also been reported to be contained within the inclusions in muscle, their relative role, if any, in the generation of the aggregates has remained unclear (2,7,13,16,18). Immunohistochemical analysis also does not allow for a determination of the relative contribution of each protein to suggest its potential importance in the pathogenesis of the inclusions.…”

“…The typical histopathological findings in muscle biopsy specimens are intracytoplasmic inclusion bodies ( Figure 1A) that reduce nitro-blue tetrazolium (NBT) and thus stain strongly with the menadione-NBT stain ( Figure 1B and Supplemental Figure 2B; supplemental material available online with this article; doi:10.1172/JCI34450DS1). The inclusions are found throughout the cytoplasm but have been observed to be frequently positioned adjacent to a myonucleus (7,(13)(14)(15)(16). Immunohistochemical analysis of muscle samples from RBM patients has suggested that the inclusions have features of aggresomes, structures formed following activation and overflow of the unfolded protein response and the ubiquitin-proteasome system, similar to inclusions seen in a number of neurodegenerative conditions (17)(18)(19).…”