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SP10: Christian A. Hübner - Glycosylation of alpha-dystroglycan in the aging and diseased muscle

Background and previous work
Age related muscle atrophy is associated with decreases in muscle mass, fiber diameter, muscle contractile function, diminished ability to respond to a hypertrophic stimulus and increased muscle cell apoptosis1. The mechanisms responsible for these changes are largely unknown. Similar alterations in muscle structure, function, and signaling have been observed in hereditary muscular dystrophies, which are caused by genetic defects of proteins of the dystrophin-glycoprotein complex like dystrophin, dystroglycans, sarcoglycans, integrins, and others2. Importantly, it has been shown that the glycosylation of different components of this complex changes in aging rodents3. Mutations in the enzyme Guanosin-Diphosphate-Mannose-Pyrophosphorylase-B (GMPPB), which catalyzes the conversion of Mannose-1-phosphate into the glycosylation substrate UDP-Mannose, result in a hypoglycosylation of alpha-dystroglycan and lead to muscular dystrophy4.
We recently identified mutations in the homologue GMPPA as causative for a rare autosomal recessive disorder characterized by achalasia (swallowing difficulties), alacrima (reduced/absent tear secretion), and mental retardation in combination with variable neurological symptoms, also known as AAMR-syndrome5. GMPPA has no enzymatic activity itself6. However, since we observed increased UDP-Mannose levels in lymphocytes of GMPPA-patients, we assume that GMPPA might be an allosteric inhibitor of GMPPB. Along this line defects in GMPPA may lead to accumulation of hyperglycosylated and potentially dysfunctional alpha-dystroglycan and other glycoproteins. To be able to further characterize the pathophysiology of AAMR-syndrome we have now established a GMPPA-knockout mouse model. Notably knockout mice develop a progressive age-dependent gait disorder. We hence hypothesize that altered glycosylation of components of the dystrophin-glycoprotein complex contributes to muscle aging.
Specific aims
We here propose to assess muscle function in aging GMPPA knockout mice. We will further study the glycosylation of alpha-dystroglycan and other components of the dystrophin-glycoprotein complex in control and GMPPA knockout mice at different ages. We will further assess whether GMPPA and GMPPB directly interact.
Working programme and collaborations
To study whether GMPPA is indeed an allosteric inhibitor of GMPPB, we will clone cDNAs for both GMPPA and GMPPB and mutant variants either labeled by Flag- or Myc-tags. We will co-express both constructs in COS-7 cells and visualize the respective subcellular localizations. We will further assess whether disease associated variants mislocalize and whether mislocalization of either variant causes mislocalization of the respective homologue. We will further investigate whether overexpressed and tagged GMPPA and GMPPB co-immunoprecipitate to prove their functional interaction. If successful we will try to verify the protein-protein interaction for endogenous GMPPA and GMPPB. We will also clone deletion constructs to resolve which parts of the respective proteins are needed for protein interaction. Finally we will assess whether bacterially expressed GMPPA and GMPPB also interact to stud whether the interaction is direct or depends on other proteins.
In parallel we will study and validate our GMPPA knockout mice as a disease model for AAMR syndrome. In particular we will quantify muscle performance in aging control and GMPPA knockout mice with the “grip test” and by forced running in an electrified treadmill. We will also perform an electromyographic analysis in aging control and GMPPA knockout mice. For this purpose we will repetitively stimulate selected motor nerves and will measure the resulting compound muscle action potentials. By concentric needle electromyography we will search for polyphasic motor units as often observed in myopathies. Selected skeletal muscles will be assessed histologically for signs of muscle degeneration like measurement of the mean cross-sectional area of muscle fibers, centralization of nuclei, which are regularly located at the periphery of the individual muscle cells, and for inflammatory cell infiltration. We will also study the localization and expression of glycosylated alpha-dystroglycan versus the protein core and other members of the dystrophin-glycoprotein complex in different skeletal muscles of aging wild-type, GMPPA knockout mice and the mouse models used in SP2 by immunofluorescence studies. Quantification of the respective proteins will be performed by western blot analysis of skeletal muscle lysates, which will also give a first hint for glycosylation changes in samples of aging and diseased mice. The molecular analysis of protein glycosylation in samples from aged and GMPPA-deficient mice will be studied by mass spectrometry.
We will collaborate with SP 1 (Glomb) and SP 2 (Horstkorte) on the analysis of protein glycosylation, with SP 8 (Simm) for the phenotypic analysis of aging mice. Protein interaction studies will be performed in close collaboration with SP 8 (Balbach) and SP 14 (Huber).

Prof. Dr. med Christian A. Hübner
Project SP10
Friedrich Schiller University of Jena
Institute of Human Genetics
Jena University Hospital
Am Klinikum 1
07747 Jena
e-mail: Christian Hübner
Homepage: AG-Hübner