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other·neurology, neuromuscular disease, mitochondrial medicine, genetics·PMC10799095

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2

Acta Neuropathologica · 15 authors, 11 centres

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This study reveals that mitochondrial dysfunction is a key feature of myotonic dystrophy type 2 (DM2), affecting respiratory chain complexes I, III, and IV, and associated with reduced mtDNA copy number and impaired mitophagy. These abnormalities were confirmed across proteomic, transcriptomic, and morphological levels in two independent patient cohorts. The findings suggest that mitochondrial pathways could represent novel therapeutic targets for DM2.

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**Background:** Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disorder caused by CCTG tetranucleotide expansions in the CNBP gene. It is characterized by proximal muscle weakness, myalgia, cataracts, cardiac abnormalities, and glucose intolerance. While research on myotonic dystrophy type 1 (DM1) has identified mitochondrial dysfunction as a potential therapeutic target, comparable molecular studies in DM2 are lacking. This study aimed to characterize the molecular pathogenesis of DM2 using a multi-level approach combining histomorphological, ultrastructural, proteomic, and transcriptomic analyses.

**Methods:** The study included two independent German cohorts: an exploratory cohort of 8 DM2 patients (4 male, 4 female; mean age at biopsy 58 ± 10.2 years) and a confirmatory cohort of 40 DM2 patients. Muscle biopsy specimens from the quadriceps femoris were analyzed. Proteomic profiling was performed on 7 DM2 samples and 5 non-diseased controls (NDC) using a label-free data-independent acquisition (DIA) workflow. Bulk RNA sequencing was conducted on frozen muscle biopsies from DM2 patients and 33 histologically normal controls. Histological and enzyme histochemical analyses (COX-SDH, SDH, COX, NADH-TR) were performed on all 48 DM2 samples and 10 NDC. Transmission electron microscopy (TEM) was used to assess ultrastructural abnormalities. Respiratory chain immunoblotting and enzymology, mtDNA copy number quantification by qPCR, and long-range PCR for mtDNA deletions were also performed.

**Key Results:** Proteomic analysis identified 3282 proteins, of which 128 were upregulated and 62 downregulated in DM2 muscle compared to NDC. Notably, 16 of the 62 downregulated proteins were involved in mitochondrial oxidative phosphorylation (OXPHOS), particularly complexes I, III, and IV. The most downregulated mitochondrial proteins included mt-CO1, mt-ND5, mt-CYB, and SLC41A3. The translational activator of cytochrome oxidase subunit I (TACO1) was also significantly downregulated. RNA sequencing showed concordant downregulation of transcripts for 13 of the 16 downregulated mitochondrial proteins. Histologically, all 48 DM2 samples exhibited features of mitochondrial dysfunction, including subsarcolemmal caps, coarse granular SDH positivity, and COX-negative fibers. TEM revealed ultrastructural abnormalities in 49% of mitochondria (n=667 mitochondria quantified), including paracrystalline inclusions (type I: 7.4%, type II: 4.8%), circular cristae (2.8%), and swollen/enlarged mitochondria (34%). mtDNA copy number was significantly reduced in DM2 patients compared to controls (p<0.05). Long-range PCR did not detect age-inappropriate mtDNA deletions. Immunoblotting showed no statistically significant differences in OXPHOS complex levels between patients and controls, but respiratory chain enzymology indicated a trend toward reduced complex I and IV activities. Immunofluorescence revealed subsarcolemmal accumulation of mitochondria co-localizing with p62 but not LC-3, suggesting impaired mitophagosome formation. Immunohistochemistry for BNIP3 showed strong subsarcolemmal staining.

**Clinical Implications:** This study provides the first comprehensive evidence that mitochondrial dysfunction is a significant component of DM2 pathophysiology, affecting complexes I, III, and IV, and involving impaired mitophagy and reduced mtDNA copy number. These findings highlight mitochondrial pathways as potential novel therapeutic targets for DM2, similar to emerging strategies in DM1. The identification of TACO1 downregulation may explain the excess COX-negative fibers observed. The lack of mtDNA deletions suggests a different mechanism from DM1. Further functional studies (e.g., Seahorse assays) are needed to confirm the metabolic impact and to develop cell culture models for therapeutic testing.

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neurology

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ORIGINAL PAPER

PMC10799095

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2

Acta Neuropathologica

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ABSTRACT

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-023-02673-y.

CITATION EXPORT

Felix Kleefeld, Rita Horvath, Iago Pinal-Fernandez et al.. Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2. Acta Neuropathologica. (2024). https://doi.org/10.1007/s00401-023-02673-y

AUTHORS

15 · 11 CENTRES

Felix Kleefeld, Rita Horvath, Iago Pinal-Fernandez, Andrew L. Mammen, Maria Casal-Dominguez, Denisa Hathazi, Sarah Melchert, Katrin Hahn, Albert Sickmann, Claudia Muselmann-Genschow, Andreas Hentschel, Corinna Preuße, Andreas Roos, Benedikt Schoser, Werner Stenzel

1grid.6363.00000 0001 2218 4662Department of Neurology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, Berlin Institute of Health (BIH), Charitéplatz 1, 10117 Berlin, Germany
2https://ror.org/013meh722grid.5335.00000 0001 2188 5934Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
3grid.420086.80000 0001 2237 2479Muscle Disease Unit, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892 USA
4grid.21107.350000 0001 2171 9311Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA
5grid.6363.00000 0001 2218 4662Department of Neurology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, Charitéplatz 1, 10117 Berlin, Germany
6https://ror.org/02jhqqg57grid.419243.90000 0004 0492 9407Leibniz-Institut Für Analytische Wissenschaften-ISAS E.V., 44139 Dortmund, Germany
7grid.6363.00000 0001 2218 4662Department of Neuropathology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, Berlin Institute of Health (BIH), Charitéplatz 1, 10117 Berlin, Germany
8grid.6363.00000 0001 2218 4662Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, Germany
9grid.5718.b0000 0001 2187 5445Pediatric Neurology, Faculty of Medicine, University Children’s Hospital, University of Duisburg-Essen, Essen, Germany
10grid.28046.380000 0001 2182 2255Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON K1H 8L1 Canada
11https://ror.org/05591te55grid.5252.00000 0004 1936 973XDepartment of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich, Germany