Citations

Abstract 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. A wide battery of complementary approaches from RNA level to high-resolution respirometry on permeabilized muscle fibers was employed. The relationship between mitochondrial respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine their clinical relevance. The study included a total of 67 patients with IBM and 45 controls. On high resolution respirometry of permeabilized muscle fibers, IBM samples exhibited reduced maximal mitochondrial 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, male patients had decreased State 3 intrinsic respiration, whereas female patients had greater intrinsic respiration under leak states. Complex I activity was decreased mainly in female patients, in whom complex II control ratio positively correlated with disease duration and severity. IBM was further associated with decreased RNA levels of all complexes, and lower protein expression of complex I, III, IV and V, likely related to the lower mtDNA content seen in IBM samples. Regarding the production of reactive oxygen species, IBM samples exhibited lower maximal H2O2 emission, accompanied by a higher total antioxidant capacity that positively correlated with disease duration in female patients. Lastly, correlation analyses suggested that impaired mitochondrial respiration, altered mitophagy, and reduced mtDNA content are interconnected in IBM and maybe of clinical significance. IBM is characterized by multifaceted, clinically relevant impairments in mitochondrial respiration. Future studies should further explore underlying pathomechanisms and the variation of mitochondrial respiration by disease stage.

Abstract Background Mono-allelic Dehydrodolichyl Diphosphate Synthase ( DHDDS) variants are associated with juvenile Parkinsonism, developmental delay and seizures. Symptoms are progressive, and various mechanisms, such as defective glycosylation, lysosomal dysfunction and cholesterol accumulation have been hypothesized to underlie disease symptoms. There is no treatment for DHDDS-related disease. Methods Patient-derived cortical forebrain organoids were created to elucidate disease mechanisms and evaluate potential treatments. In these neuronal models, glycosylation, lipidomics, proteomics, cholesterol/ganglioside accumulation, mitochondrial function and electrophysiological activity were assessed. Finally, we investigated the effects of nicotinamide mononucleotide (NMN), identified through a yeast-based drug screen, in neuronal cell models and in six patients in an off-label, N-of-1, observational series. Results DHDDS-patient derived organoids showed visual signs of degeneration after four months of culturing. This was accompanied by significant cholesterol accumulation in astrocytes, decreased mitochondrial respiration and loss of deep-layer neurons. In addition, we identified glycosylation abnormalities, showing for the first time that glycosylation in human tissue is affected by monoallelic DHDDS variants. Proteomic analysis revealed altered protein expression of proteins involved in lipid metabolism, cytoskeletal organization and neuronal development. We found that oral Nicotinamide Mononucleotide supplementation led to significant improvement in mitochondrial respiration and electrophysiological parameters in organoids, concurring with clinical improvements in all of the treated patients, particularly regarding their ataxia and tremor. Conclusion Our findings reveal a progressive phenotype in DHDDS-patient-derived brain organoids, with mitochondrial dysfunction and astrocyte-specific metabolic alterations contributing to disease pathology. Notably, NMN treatment led to clinical improvements in patients with heterozygous DHDDS variants, highlighting its potential as a therapeutic strategy.

Introduction: Ubiquinol–cytochrome c reductase core protein II (UQCRC2) encodes a core subunit of the mitochondrial electron transport chain (ETC) complex III (CIII). Biallelic pathogenic variants in UQCRC2 have been associated with mitochondrial disease characterized by lactic acidosis, developmental delay, hepatopathy, and episodic metabolic decompensation. Methods: We reviewed the biochemical phenotypes of 14 individuals possessing UQCRC2 variants, including two novel cases. We performed biochemical studies of mitochondrial respiration and oxidative phosphorylation (OXPHOS) complex measurements in patient-derived fibroblasts. Results: We report reduced CIII activity in a majority of individuals possessing variants in UQCRC2, as well as biochemical findings consistent with impaired mitochondrial energy metabolism, though impairments in mitochondrial respiration were variable. The two previously unreported, unrelated patients possessing the likely pathogenic missense variant c.361T>C, p.Tyr121His in UQCRC2 in trans with a 16p12.2 microdeletion encompassing UQCRC2 showed milder phenotypes, less severe metabolic decompensations, and no long-term neurological impairments. Both individuals display reduced CIII activity and mitochondrial respiratory dysfunction. Discussion: These data expand the current understanding of genotypes associated with UQCRC2-associated mitochondrial disease to include the novel 16p12.2 microdeletion. These data also highlight the consistent biochemical phenotype associated with UQCRC2-associated mitochondrial disease, and the need for consistent biochemical and respiratory assessment of individuals possessing UQCRC2 variants to further our understanding of this phenotype.

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.

Abstract Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase ( cis- PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, Dhdds R37H+/- strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. Dhdds R37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used “off-label” to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in Dhdds R37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis- PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

Abstract The lack of a molecular understanding of mood disorders has impeded progress in diagnosis and treatment. Glycosylation may provide insights into the complex mechanisms underlying these conditions. We conducted N-glycoproteomic analysis on dorsolateral prefrontal cortex samples from individuals with major depressive disorder (MDD) and bipolar disorder (BD), in depressive or manic states at death. Additionally, we examined depression prevalence in congenital disorders of glycosylation (CDG) through a literature review and assessment of 110 CDG patients. Glycoproteomic analysis revealed a significant increase in protein glycosylation in individuals with MDD relative to both controls and individuals with BD. Depression prevalence was lower in our pediatric and adult cohort of individuals with CDG. These results suggest brain glycosylation changes may play a role in mood disorder pathology and highlight the distinct biology of unipolar and bipolar depression. Our findings propose that impaired glycosylation may confer resilience to depression, offering potential therapeutic insights.

Abstract Congenital disorders of glycosylation (CDG) are a group of neurogenetic conditions resulting from disruptions in the cellular glycosylation machinery. The majority of CDG patients have compound heterozygous pathogenic variants in the phosphomannomutase 2 ( PMM2) gene. Individuals with PMM2-CDG exhibit multi-systemic symptoms, prominently featuring neurological deficits with nearly all patients exhibiting cerebellar hypoplasia and ataxia. To overcome embryonic lethality caused by whole body knock-out of Pmm2 and mimic patient-related compound heterozygous pathogenic variants, we paired a Pmm2 flox allele ( Pmm2 fl ) with a catalytically inactive knock-in allele ( Pmm2 R137H ), commonly present in PMM2-CDG patients. Mice with post-mitotic loss of PMM2 from neurons or astrocytes are indistinguishable from unaffected littermates, including in a broad battery of neurological assessments. In contrast, removal of PMM2 from embryonic neural precursor cells leads to cerebellar hypoplasia, ataxia, seizures, and early lethality. Comprehensive multi-omics profiling, including metabolomics, glycomics, single-cell transcriptomics, proteomics, and glycoproteomics, reveal widespread molecular disturbances throughout the brain, with the cerebellum showing the most pronounced disruption. These findings highlight the heightened dependency of the developing cerebellum on intact N-glycosylation, aligning with clinical observations in PMM2-CDG patients. Importantly, glycoproteomic alterations identified in our mouse model are corroborated in PMM2-CDG patient post-mortem cerebellar tissue, underscoring the translational relevance of our findings and implicating impaired synaptic transmission as a key pathogenic mechanism.

Inherited metabolic disorders (IMDs) are genetic disorders that occur in as many as 1:2500 births worldwide. Nevertheless, they are quite rare individually and even more rare is the co-occurrence of two IMDs in one individual. To better understand the metabolic cross-talk between glycosylation changes and deficient energy metabolism, and its potential effect on outcomes, we evaluated patient fibroblasts with likely pathogenic variants in PGM1 and pathogenic variants in NDUFA13 derived from a patient who passed away at 16 years of age. The patient presented with characteristic of PGM1-CDG including bifid uvula, muscle involvement, abnormal glycosylation in blood, and elevated liver transaminases. In addition, hearing loss, seizures, elevated plasma and CSF lactate and a Leigh-like MRI brain pattern were present, which are commonly associated with Leigh syndrome. PGM1-CDG has been reported in about 70 individuals, while NDUFA13 deficiency has so far only been reported in 13 patients. As abundant energy is essential for glycosylation, and both PGM1 and NDUFA13 are linked to energy metabolism, we sought to better understand the underlying biochemical cause of the patient’s clinical presentation. To do so, we performed extensive investigations including tracer metabolomics, lipidomics and enzymatic studies on the patient’s fibroblasts. We found a profound depletion of UDP-hexoses, consistent with PGM1-CDG. Complex I enzyme activity and mitochondrial function were also impaired, corroborating complex I deficiency and Leigh syndrome. Further, lipidomics analysis showed similarities with both PGM1-CDG and OXPHOS-deficient patients. Based on our results, the patient was diagnosed with both PGM1-CDG and Leigh syndrome. In summary, we present the first case of combined CDG and Leigh syndrome, caused by (likely) pathogenic variants in PGM1 and NDUFA13, and underline the importance of considering the synergistic effects of multiple disease-causing variants in patients with complex clinical presentation, leading to the patient’s early demise.