Arnaud Bruneel

Apical radial glial cells (aRGs) are highly polarized epithelial cells that drive human cortical development through tightly regulated lineage progression. A distinctive feature of aRGs is their non-canonical Golgi organization, raising the possibility that specialized intracellular trafficking and secretory processes contribute to progenitor behaviour. However, how Golgi architecture contributes to human corticogenesis remains poorly understood. Here, we investigate this question in the context of COG5 deficiency, a rare congenital disorder of glycosylation associated with neurodevelopmental defects, using patient-derived fibroblasts and brain organoids, N-glycomics, glycolipidomics, and single-cell transcriptomics. We show that COG5 deficiency disrupts Golgi organization and impairs Golgi-to-ER retrograde trafficking. At the neurodevelopmental level, COG5-deficient organoids display altered ventricular organization and reduced basal progenitor abundance. Single-cell transcriptomic analysis reveals rewired developmental trajectories characterized by premature differentiation from apical progenitors with activation of neuronal pathways. Despite the absence of major abnormalities in plasma glycoproteins glycosylation, glycolipidomic analysis identified marked defects in glycosphingolipid maturation within patient-derived brain organoids, indicating selective impairment of neural lipid glycosylation. Transcriptomic and biochemical analyses further revealed dysregulation of developmental pathways, including reduced Notch signalling and altered localization of both cleaved Notch1 and the Golgi-associated protein ACBD3. Consistent with impaired progenitor maintenance, COG5-deficient organoids displayed altered cleavage plane orientation in apical progenitors. Together, our findings suggest a link between Golgi organization, intracellular trafficking, membrane glycosylation, and lineage progression in human neural progenitors, identifying Golgi architecture as an important regulator of apical radial glial cell biology and cortical development.

Background and Aims: To develop an assay to measure red blood cell (RBC) sorbitol dehydrogenase (SORD) activity for fast laboratory screening of SORD‐related neuropathies, and to provide additional phenotypic arguments in case of ambiguous genetic results. Methods: Hemolysates were incubated in the presence of fructose and NADH to produce sorbitol and NAD ⁺ . The decrease in absorbance at λ = 340 nm was monitored throughout the course of the reaction. Results were expressed as % activity compared to a within‐run control. A cohort of 38 individuals was evaluated over 13 runs: 13 individuals served as within‐run controls, 3 were diagnosed with SORD deficiency, 2 were highly suspected of having SORD deficiency, 1 exhibited incidental genomic findings in the SORD gene, and 19 presented with neuropathy or myopathy of undetermined etiology. Results: RBC SORD activity was significantly reduced in individuals diagnosed with SORD deficiency when compared to within‐run controls, whereas in contrast, individuals without SORD deficiency did not exhibit this reduction. A comparison of these two groups showed that activities were significantly lower in deficient individuals ( p = 0.00006). A test cutoff of < 30% yielded 100% sensitivity and specificity to detect deficient individuals. Interpretation: This test provides a useful alternative to measuring plasma or urine sorbitol with mass spectrometry, allowing clinicians to directly evaluate SORD activity in RBC. As a result, it enables quick and convenient screening for SORD‐related neuropathies during standard clinical procedures.

ATP6AP2 splicing variants cause syndromic X‐linked intellectual disability Hedera type (XPDS; OMIM#300423) and X‐linked parkinsonism with spasticity (MRXSH; OMIM#300911). Alternatively, ATP6AP2 missense variants lead to hepatopathy, immunological abnormalities, cutis laxa and only mild intellectual disability with N‐/O‐glycosylation defects (ATP6AP2‐CDG; OMIM#301045). The disparity between neurological and hepatic ATP6AP2‐related disease entities is an ongoing puzzle. We aimed to investigate whether patients with an isolated neurological presentation of ATP6AP2‐related disease, consistent with XPDS/MRXSH, also have abnormal glycosylation biomarkers, potentially implicating this as part of the pathological mechanism. We identified three males and one female from three families with ATP6AP2 splicing variants and ID/DD, epilepsy, axial hypotonia, axonal neuropathy and microcephaly; the heterozygous female has a milder phenotype. RNA‐Seq in patient‐derived fibroblasts validated defective splicing, correlated with lowered ATP6AP2 protein levels in fibroblasts alongside glycosylation abnormalities. We describe defective glycosylation alongside ATP6AP2 splicing variants in four patients, including the first female with ATP6AP2 ‐related disease. This connects more closely the phenotypes of XPDS/MRXSH and ATP6AP2‐CDG and indicates that abnormal glycosylation markers may be a consistent feature of splicing variants, and potentially part of the pathological mechanism underlying ATP6AP2‐related disease caused by abnormal splicing. We also provide additional evidence that neurodevelopment is uniquely sensitive to the gene dosage of ATP6AP2, linked to the isolated neurological phenotype found in patients with splice variants and the attenuated, but still severe, phenotype of the female in our study. Glycosylation defects can be found in “splicing” forms of ATP6AP2‐related diseases, bridging the gap between XPDS, MRXSH and ATP6AP2‐CDG.

Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is a chronic demyelinating neuropathy with deposits of IgM and sural nerve fiber demyelination. While growing evidence supports the critical role of IgG glycosylation in autoimmune diseases, IgM glycosylation profiles markedly differ from those of IgG but are largely neglected. The aim of this study was to characterize IgM N-glycosylation in patients with anti-MAG neuropathy and its involvement in anti-MAG pathogenicity. IgM antibodies were isolated from patients with anti-MAG neuropathy (n = 17), asymptomatic patients with monoclonal gammopathy of undetermined significance (n = 8), and healthy donors ([HDs], n = 6). N-glycan analysis was performed using mass spectrometry. Binding to myelin-associated glycoprotein (MAG), complement (C1q), and IgM Fc receptors (Fcα/μR and DC-SIGN) was compared using ELISA between anti-MAG and MGUS IgM, before and after N-deglycosylation/desialylation. Finally, we assessed how IgM N-glycosylation influences cytokine production by monocyte-derived macrophages by measuring cytokine levels in culture supernatants. Anti-MAG IgM exhibited a unique glycosylation pattern, dominated by fucosylated, monosialylated N-glycan with a bisecting N-acetyl glucosamine (N-glycan 12), representing 48.5% of the total N-glycan pool, compared with 27.3% in MGUS IgM and 35.6% in HD IgM. We showed that deglycosylation and desialylation significantly reduced anti-MAG activity and C1q binding (average % of decrease 58.3 ± 18.8, p < 0.01, and 40.0 ± 19.9%, p < 0.05, respectively). Furthermore, anti-MAG IgM binding to C1q was significantly higher than that of MGUS IgM and HD IgM (p < 0.0001 and p < 0.001, respectively). Compared with MGUS IgM, anti-MAG IgM also significantly increased the production of proinflammatory cytokines IL-1, IL-6, IL-8, TNF-α, and IFN-Υ by macrophages, in a glycan-dependent manner (p < 0.01 to p < 0.001), with IL-8 being particularly elevated. Finally, we found that anti-MAG IgM bound more strongly Fcα/μ receptor and DC-SIGN compared with MGUS IgM (p < 0.05 and p = 0.06). This study uncovered a unique N-glycosylation pattern of anti-MAG IgM, crucial for its interaction with MAG and binding to C1q. Moreover, anti-MAG IgM increased the macrophage cytokine production, driven by their glycosylation. The increased IL-8 expression and anti-MAG IgM binding to C1q might open 2 potential therapeutic avenues: inhibiting IL-8 activity or targeting the complement pathway. In addition, the glycosylation and C1q binding of anti-MAG IgM could serve as biomarkers for monitoring this neuropathy.

Congenital disorders of glycosylation (CDG) constitute an increasing group of inborn metabolic disorders, with more than 170 described diseases to date. A disturbed glycosylation process characterizes them, with molecular defects localized in distinct cell compartments. In CDG, N-glycosylation, O-glycosylation, glycosylation of lipids (including phosphatidylinositol) as well as the glycosaminoglycan synthesis can be affected. Owing to the importance of glycosylation for the function of concerned proteins and lipids, glycosylation defects have diverse clinical consequences. CDG affected individuals often present with a non-specific multivisceral syndrome including neurological involvement, intellectual disability, dysmorphia, and hepatopathy. As CDG are rare diseases frequently lacking distinctive symptoms, biochemical and genetic testing bear important and complementary diagnostic roles. After an introduction on glycosylation and CDG, we review current biomarkers and analytical techniques in the field. Furthermore, we illustrate their interests in the follow-up of proven therapeutic approaches including D-mannose in MPI-CDG, D-galactose in PGM1-CDG, and manganese (MnSO₄) in TMEM165-CDG.

GRASP65 is a Golgi-associated peripheral protein encoded by theGORASP1gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant ofGORASP1(c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.