Smith, D. Y. | Search bioRxiv

bioRxiv2025

ALG13 loss-of-function alters glycosylation, impairs neuronal maturation, and drives network hypoactivity in a cortical organoid model of CDG

BackgroundCongenital disorders of glycosylation (CDGs) are a group of rare metabolic diseases recognized for their neurological presentations, including developmental delay and seizures. However, the link between glycosylation defects and cortical brain network pathology remains elusive. MethodsTo address this unmet need, we generated iPSC derived human cortical organoids (hCOs) for ALG13-CDG, which is the second most common CDG that is also X-linked. To comprehensively understand the impact of glycosylation defects on cortical pathology in CDG, we combined electrophysiological recordings using multi-electrode arrays (MEA) with comprehensive molecular profiling via multiomics, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was also evaluated in both iPSCs and organoids. ResultsALG13-CDG hCOs revealed reduced glycosylation of proteins critical for extracellular matrix (ECM), neuronal migration, lipid metabolism, calcium ion homeostasis, and neuronal excitability. Dysregulation in related pathways was corroborated by proteomics and scRNA-seq, which also showed altered communication patterns in these pathways. Trajectory analysis revealed an inversion in neuronal development, with early inhibitory and delayed excitatory development, indicating an excitatory and inhibitory (E/I) imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst dynamics, and shorter axonal extensions. Despite this, transcriptomic and proteomic data revealed upregulation of excitatory receptors suggesting latent hyperexcitability. Altered lipid and sugar (GlcNAc) metabolism and skewed X-inactivation were also observed. ConclusionsOur study provides the first evidence of glycosylation defects in an ALG13-CDG human cortical organoid (hCO) model and links these defects to disrupted neuronal developmental trajectories and dysregulation of key pathways essential for brain function. We identify mistimed neuronal maturation and an excitatory/inhibitory (E/I) imbalance as early drivers of network hypoactivity and immature burst dynamics, with downstream compensatory hyperexcitability that may contribute to seizure susceptibility. While specific to ALG13-CDG, these mechanisms likely extend to other glycosylation disorders with overlapping neurological features. This work offers new mechanistic insight into cortical dysfunction associated with impaired protein glycosylation and highlights potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2021

Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive distal myopathy with rimmed vacuoles that typically manifests in late adolescence/early adulthood. GNE encodes an enzyme that is the rate-limiting step in sialic acid biosynthesis which is necessary for proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study we aimed to address this knowledge gap by querying underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem cell (iPSC) model. Muscle and skin biopsies were acquired from two patients with GNE myopathy that presented with distinct histopathological features. Control and patient-specific iPSCs were derived from skin fibroblasts and differentiated down a skeletal muscle lineage in a three-stage process analogous to muscle development and muscle regeneration. Initial studies revealed: 1) the ability of patient-derived GNE iPSC clones to recapitulate key characteristics of the human pathology including TDP-43 accumulation and evidence of dysregulated autophagy, and 2) a striking defect in myogenic progression of the more severe GNE iPSC clone. Single-cell RNA sequencing time course studies were then performed to more rigorously explore myogenesis defects. Cluster-based bioinformatics analyses revealed clear differences between control and GNE iPSC-derived muscle precursor cells (iMPCs). On a transcriptional level, late stage GNE iMPCs resembled that of early stage control iMPCs, confirming stalled myogenic progression on a molecular level. Comparative expression and pathway studies revealed EIF2 signaling as a top signaling pathway altered in GNE iMPCs. In summary, we report a novel in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a likely novel contributing mechanism to the etiology of GNE myopathy. SUMMARY STATEMENTDevelopment of a novel cell-based model of GNE myopathy, utilizing GNE patient-derived samples, which recapitulates human disease characteristics, uncovered myogenic differentiation defects, and can elucidate possible mechanistic contributors to the disease.

developmental biology↗