Breyer, M. | bioRxiv Science

bioRxiv2025

Axon guidance deficits in a human sensory neuron model of Fabry disease

Fabry disease (FD) is a rare genetic galactosidase alpha (GLA) gene associated lysosomal disorder caused by alpha-galactosidase A (AGAL) deficiency, leading to sphingolipid (globotriaosylceramide, Gb3) accumulation in multiple tissues. Burning pain due to small fiber neuropathy is an early symptom with great impact on health- related quality of life. The pathophysiological role of Gb3 accumulations in sensory neurons of the dorsal root ganglia is incompletely understood. We have differentiated induced pluripotent stem cells of an isogenic GLA knockout line (p.S364del, hemizygous) and its healthy control into sensory neurons to model FD in vitro. We have compared both lines on transcriptional and proteomic level and investigated the effects of AGAL enzyme supplementation. FD sensory neurons showed dysregulation of disease-related pathways, including axon guidance at both RNA and protein level and microfluidic assays revealed shorter neurite length. While AGAL did not restore the transcriptomic state, it reduced Gb3 accumulation and lowered protein ephrin 5A and glycoprotein M6A level. These findings highlight axon guidance alterations in an isogenic human FD sensory model, with potential implications for early central and peripheral innervation in small fiber neuropathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/673441v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@199cd19org.highwire.dtl.DTLVardef@682521org.highwire.dtl.DTLVardef@15da60borg.highwire.dtl.DTLVardef@1404c10_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model

Acral burning pain triggered by fever, thermal hyposensitivity, and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide (Gb3). To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via CRISPR/Cas9 gene editing. We subjected iPSC to targeted peripheral neuronal differentiation and observed intra-lysosomal Gb3 accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in Fabry cell lines as compared to the healthy control. Moreover, we demonstrate a drastic increase in Fabry sensory neuron Ca2+ levels at 39{degrees}C mimicking clinical fever (p < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons obtained from Fabry patients compared to healthy control cells (p < 0.001). Linear-Nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed jam of mitochondrial trafficking at sphingolipid accumulations within Fabry sensory neurites utilizing a click-chemistry approach together with mitochondrial dysmorphism compared to healthy control cells. We pioneer insights into the cellular mechanisms contributing to pain, thermal hyposensitivity, and denervation in Fabry small fibre neuropathy, and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

neuroscience↗