Cardiovascular disease is one of the leading causes of death in developed countries. Because the incidence increases exponentially in the aging population, aging is a major risk factor for cardiovascular disease. Cardiac hypertrophy, fibrosis and inflammation are typical hallmarks of the aged heart. The molecular mechanisms, however, are poorly understood. Because glycosylation is one of the most common post-translational protein modifications and can affect biological properties and functions of proteins, we here provide the first analysis of the cardiac glycoproteome of mice at different ages. Western blot as well as MALDI-TOF based glycome analysis suggest that high-mannose N-glycans increase with age. In agreement, we found an age-related regulation of GMPPB, the enzyme, which facilitates the supply of the sugar-donor GDP-mannose. Glycoprotein pull-downs from heart lysates of young, middle-aged and old mice in combination with quantitative mass spectrometry bolster widespread alterations of the cardiac glycoproteome. Major hits are glycoproteins related to the extracellular matrix and Ca²⁺-binding proteins of the endoplasmic reticulum. We propose that changes in the heart glycoproteome likely contribute to the age-related functional decline of the cardiovascular system.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.

Guanosine diphosphate-mannose pyrophosphorylase B (GMPPB) catalyzes the conversion of mannose-1-phosphate and GTP to GDP-mannose, which is required as a mannose donor for the biosynthesis of glycan structures necessary for proper cellular functions. Mutations in GMPPB have been associated with various neuromuscular disorders such as muscular dystrophy and myasthenic syndromes. Here, we report that GMPPB protein abundance increases during brain and skeletal muscle development, which is accompanied by an increase in overall protein mannosylation. To model the human disorder in mice, we generated heterozygous GMPPB KO mice using CIRSPR/Cas9. While we were able to obtain homozygous KO mice from heterozygous matings at the blastocyst stage, homozygous KO embryos were absent beyond embryonic day E8.5, suggesting that the homozygous loss of GMPPB results in early embryonic lethality. Since patients with GMPPB loss-of-function manifest with neuromuscular disorders, we investigated the role of GMPPB in vitro. Thereby, we found that the siRNA-mediated knockdown of Gmppb in either primary myoblasts or the myoblast cell line C2C12 impaired myoblast differentiation and resulted in myotube degeneration. siRNA-mediated knockdown of Gmppb also impaired the neuron-like differentiation of N2A cells. Taken together, our data highlight the essential role of GMPPB during development and differentiation, especially in myogenic and neuronal cell types.

Three articles assessed the neurodegenerative diseases Alzheimer (AD) and Parkinson (PD) as well as key regulators of ageing. Varshavskaya et al., 2024 investigated how PTMs of βamyloid (Aβ) may affect AD. In AD, Aβ plaques accumulate within the brain and hence serve as a biomarker for this disorder. The authors provided evidence for phosphorylated Aβ 42 as well as isomerized Aβ 42 to cross an in-vitro blood-brain barrier more efficiently than unmodified Aβ 42 , and reported a different mechanism of transport. These findings could be significant for understanding Alzheimer's disease (AD) pathogenesis and treatment, and may prove valuable in the search for biomarkers. Jonischkies et al., 2024 stressed the nuclear DBF2-related (NDR) family of serine-threonine AGC kinases as essential regulators of ageing by underlining their functions, such as autophagy and inflammatory cytokine regulation, nutrient, AMPK or insulin signaling, and dysfunctions in the context of ageing. Luo et al., 2024 highlighted recent findings on major PTMs, such as ubiquitination, phosphorylation, SUMOylation, acetylation, or Snitrosylation, on mitochondrial dysfunction, a central factor in PD pathogenesis. Moreover, they discussed the potential of proteins harboring PTM sites, such as α-synuclein or VPS35, as biomarkers for PD. In agreement with mitochondrial changes upon PTM alterations, Liu et al., 2024 showed that chronic intermittent hypoxia (CHI) alters hippocampal protein acetylation in mice. The majority of the affected proteins were involved in mitochondrial processes including oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. Mice under CHI treatment showed cognitive impairment, hippocampal lesions, glial cell activation, inhibited neurogenesis and induced inflammation. Similarly, hippocampal lesions, plaques, and astrocytic gliosis are observed in prion disease. Bizingre et al., 2024 discussed recent findings on the impact of altered PTMs on the prion protein (PrPC), its function, and its conversion into the pathogenic variant. They also explored prion-related downstream factors as potential drug targets. Two articles assessed the role of ubiquitination for protein function and stability in the context of neuronal development. Day et al., 2024 revealed that the Anaphase Promoting Complex (APC/C), an E3 ubiquitin ligase, regulates primary neurite formation and protein levels of the deubiquitinase ubiquitin specific peptidase 1 (USP1). Notably, SUMOylation of APC/C did not affect USP1 levels and neuron morphology. Franzka et al, 2024 assessed ubiquitination of GDP-mannose pyrophosphorylase B (GMPPB). GMPPB is important for generating GDPmannose, which serves as a mannose donor for glycosylation. The authors showed that ubiquitination of GMPPB neither affects its stability nor its interaction with GMPPA, but modulates its enzymatic activity. Moreover, they disclosed that patient mutations could alter GMPPB ubiquitination. Thus, ubiquitination provides another level to regulate GMPPB activity and mannosylation. Notably, loss of GMPPB results in embryonic lethality as shown by Schurig et al., 2024. Knockdown of GMPPB disrupted myoblast differentiation leading to myotube degeneration, and impaired neuron-like differentiation in N2A cells. In accordance, the authors reported that GMPPB protein abundance increased during brain and skeletal muscle development, which was accompanied by an increase in overall protein mannosylation. Another protein implicated in glycosylation disorders is the Inositol polyphosphate 5phosphatase K (INPP5K), a phosphatase of phosphoinositides (PIs). Manzolillo et al., 2024 demonstrated that INPP5K expression increases during brain development, and its knockdown impaired neuronal-like differentiation of N2A cells, while disrupting protein glycosylation. The final article in this Research Topic, by Majchrzak et al., 2024, explored the interactions between muscle stem cells and their immediate niche, the regulatory role of post-translational modifications (PTMs), and how these factors influence quiescence, activation, and selfrenewal, particularly in the context of aging and disease.In summary, this Research Topic highlighted the critical role of PTMs for nervous system and muscle function, as well as their interconnection during development, disease and ageing.PTMs are crucial for biological processes contributing to healthy organ function. Protein post-translational modifications (PTMs), such as phosphorylation (P), acetylation (Ac), SUMOylation (SUMO), S-nitrosylation (Nitro), ubiquitination (Ub) and glycosylation (Glyco), affect a wide range of cellular and biological functions as depicted in this cartoon. Perturbations lead to severe consequences for the normal function of the brain and other organs, such as muscle. Created in BioRender. Hübner, C. (2024) BioRender.com/j49w898.

GDP-mannose pyrophosphorylase B (GMPPB) loss-of-function is associated with muscular dystrophy and variable additional neurological symptoms. GMPPB facilitates the catalytic conversion of mannose-1-phosphate and GTP to GDP-mannose, which serves as a mannose donor for glycosylation. The activity of GMPPB is regulated by its non-catalytic paralogue GMPPA, which can bind GDP-mannose and interact with GMPPB, thereby acting as an allosteric feedback inhibitor of GMPPB. Using pulldown, immunoprecipitation, turnover experiments as well as immunolabeling and enzyme activity assays, we provide first direct evidence that GMPPB activity is regulated by ubiquitination. We further show that the E3 ubiquitin ligase TRIM67 interacts with GMPPB and that knockdown of TRM67 reduces ubiquitination of GMPPB, thus reflecting a candidate E3 ligase for the ubiquitination of GMPPB. While the inhibition of GMPPB ubiquitination decreases its enzymatic activity, its ubiquitination neither affects its interaction with GMPPA nor its turnover. Taken together, we show that the ubiquitination of GMPPB represents another level of regulation of GDP-mannose supply.