Kozicz, T.

ABSTRACT Polyprenal reductase is an enzyme encoded by the SRD5A3 gene, which is involved in the synthesis of dolichol from polyprenol. Dolichol serves as a carrier for glycan precursors or monosaccharides in N‐linked glycosylation. Pathogenic variants in SRD5A3 can result in a congenital disorder of glycosylation (CDG), SRD5A3‐CDG, which is inherited in an autosomal recessive manner. Most plasma proteins are glycosylated and changes in the glycosylation of several glycoproteins are associated with pathological consequences. Despite the critical role of SRD5A3 in glycosylation, the impact of its deficiency on the glycosylation of serum proteins remains largely unexplored. In this study, we used tandem mass tag‐based multiplexed quantitative approach to analyze serum N‐glycoproteomics and proteomics in SRD5A3‐CDG patients and controls. We quantified 2200 serum N‐glycopeptides from 359 N‐glycosites from 204 serum proteins. Extensive hypoglycosylation of serum proteins was observed in patients, with 245 of 291 altered glycopeptides decreased in SRD5A3‐CDG. Altered glycopeptides included those derived from haptoglobin, plasma serine protease inhibitor, alpha‐1‐B glycoprotein, alpha‐2‐macroglobulin, and ceruloplasmin. Some of these proteins have previously been reported to be associated with liver dysfunction, anemia, and coagulopathy, which could underlie similar clinical features observed in SRD5A3‐CDG patients. Overall, our study provides novel insights into alterations in the glycosylation status of specific serum proteins in SRD5A3‐CDG. Some of these alterations could be further pursued to develop glycopeptide‐based biomarkers as the current diagnosis of SRD5A3‐CDG by screening assays remains challenging. In addition, knowledge of altered glycoproteins could enhance our understanding of the disease spectrum and potentially unveil additional therapeutic avenues.

Abstract Background Oxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. Methods A wide battery of complementary tests from RNA level to high-resolution respirometry on permeabilized muscle fibers was performed. The relationship between respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine the clinical significance of the findings. Results The study included a total of 67 patients with IBM and 45 controls. IBM muscle tissue exhibited reduced maximal respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, males had decreased State 3 intrinsic respiration, whereas females had greater intrinsic respiration in leak states. Complex II control ratio strongly correlated with disease duration and severity only in females. IBM was associated with decreased RNA and protein expression of OXPHOS complexes. Complex I activity was decreased mainly in females. IBM samples exhibited lower maximal H 2 O 2 emission, accompanied by a higher total antioxidant capacity that correlated with disease duration in females. In IBM, there was decreased mtDNA content, and impaired mitophagy, both of which strongly correlated with respirometry measures and markers of disease severity, indicating these pathways are likely interconnected and of clinical significance. Conclusion IBM is characterized by multilevel impairments in mitochondrial coupling efficiency, revealing several potential therapeutic targets to improve mitochondrial fitness, while accounting for sex-specific differences.

Background: ALG13-CDG is an X-linked N-linked glycosylation disorder caused by pathogenic variants in the glycosyltransferase ALG13, leading to severe neurological manifestations. Despite the clear CNS involvement, the impact of ALG13 dysfunction on human brain glycosylation and neurodevelopment remains unknown. We hypothesize that ALG13-CDG causes brain-specific hypoglycosylation that disrupts neurodevelopmental pathways and contributes directly to cortical network dysfunction. Methods: We generated iPSC-derived human cortical organoids (hCOs) from individuals with ALG13-CDG to define the impact of hypoglycosylation on cortical development and function. Electrophysiological activity was assessed using MEA recordings and integrated with multiomic profiling, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was evaluated in both iPSCs and hCOs. Results: ALG13-CDG hCOs showed reduced glycosylation of proteins involved in ECM organization, neuronal migration, lipid metabolism, calcium homeostasis, and neuronal excitability. These pathway disruptions were supported by proteomic and scRNA-seq data and included altered intercellular communication. Trajectory analyses revealed mistimed neuronal maturation with early inhibitory and delayed excitatory development, indicating an E/I imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst structure, and shortened axonal projections, while transcriptomic and proteomic signatures suggested emerging hyperexcitability. Altered lipid and GlcNAc metabolism, along with skewed X-inactivation, were also observed. Conclusions: Our study reveals that ALG13-CDG is a disorder of brain-specific hypoglycosylation that disrupts key neurodevelopmental pathways and destabilizes cortical network function. Through integrated multiomic and functional analyses, we identify early network hypoactivity, mistimed neuronal maturation, and evolving E/I imbalance that progresses to compensatory hyperexcitability, providing a mechanistic basis for seizure vulnerability. These findings redefine ALG13-CDG as disorders of cortical network instability, offering a new framework for targeted therapeutic intervention.