Background & Aims
In Fabry disease (FD), mutations in GLA encoding for the enzyme alpha-galactosidase A (GLA) lead to accumulations of globotriaosylceramide (Gb3) in various cell types.1 FD is associated with small fiber neuropathy2 (SFN) characterized by triggerable pain, thermal hyposensitivity, and skin denervation.3 However, the underlying pathophysiology of SFN in FD is poorly understood. The inaccessibility of human sensory neurons, which play a pivotal role in pain perception, poses a substantial challenge when attempting to study the underlying pathophysiology of SFN in FD. Our objective was to establish a patient-derived in vitro model of FD using induced pluripotent stem cells (iPSC) and differentiate these into sensory neurons (iSN), which maintain the distinctive feature of cellular Gb3 accumulations. Via this model, we aimed to provide insights into the pathophysiology of SFN in FD.
Methods
Dermal fibroblasts derived from skin punch biopsies of two patients with FD (FD1, FD2) and one healthy control (Ctrl) were reprogrammed into iPSC using an mRNA-based protocol.4 Additionally, we generated an isogenic control line (ISO-FD) via CRISPR/Cas9 gene editing.5 iSN were differentiated via small molecule inhibition.6 Gene and protein expression levels of iSN markers were validated via qPCR and immunocytochemistry (ICC). Gb3 positive iSN were quantified after agalsidase-beta7 (AGAL) incubation. We further determined gene expression of voltage-gated ion channels using a micro-array. On functional level, we performed calcium imaging and whole-cell patch-clamp including recordings at room temperature (RT) and at 39°C. We analyzed action potential parameters, and activation and inactivation curves of voltage-gated sodium channels (NaV). Furthermore, we characterized excitability of single neurons via Linear-Nonlinear (LN) and Generalized Linear point process (GLM) models.
Results
Expression of iSN-specific markers after differentiation was validated for all cell lines. While Gb3 was absent in Ctrl iSN, iSN from all FD lines showed dense intra-lysosomal Gb3 accumulations. AGAL incubation decreased Gb3 deposits in FD1 (p < 0.001) and FD2 (p < 0.01). Expression of several pain-associated ion channels was increased in iSN from FD1, whereas expression in FD2 and ISO-FD was decreased compared to Ctrl. We found elevated neuronal calcium levels (p < 0.001) accompanied by neurite thinning (p < 0.001) in FD1 and FD2 iSN compared to Ctrl. Firing frequency, half-width and rising slope of action potentials did not differ between the cell lines but increased at 39°C compared to RT (p < 0.001). NaV V1/2 steady-state inactivation was decreased in ISO-FD at 39°C compared to Ctrl (p < 0.001), whereas activation was similar in all cell lines. Furthermore, LN and GLM models revealed that FD1 iSN were more selective for the spike-triggered average (STA) compared to Ctrl (p < 0.05).
Conclusions
We established a patient-derived neuronal in vitro model of FD and show that the disease phenotype can be maintained during reprogramming of iPSC and differentiation into iSN. We further conducted pilot experiments to investigate the underlying pathophysiology of SFN in FD. Pilot findings of elevated neuronal calcium levels at heat, thinning of neurites, and electrical hypoexcitability suggest a potential connection to neuropathic pain triggered by heat,8 skin denervation, and thermal hyposensitivity9 in FD, respectively. Our in vitro FD model paves the way for future studies of underlying disease mechanisms and serves as a basis to develop new treatment options.
References
1.Germain DP. Fabry disease. Orphanet J Rare Dis 2010;5:30.
2.Schiffmann R. Fabry disease. Pharmacol Ther 2009;122:65-77.
3.Üçeyler N, Ganendiran S, Kramer D, Sommer C. Characterization of pain in fabry disease. Clin J Pain 2014;30:915-920.
4.Poleganov MA, Eminli S, Beissert T, et al. Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion. Hum Gene Ther 2015;26:751-766.
5.Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8:2281-2308.
6.Eberhardt E, Havlicek S, Schmidt D, et al. Pattern of Functional TTX-Resistant Sodium Channels Reveals a Developmental Stage of Human iPSC- and ESC-Derived Nociceptors. Stem Cell Reports 2015;5:305-313.
7.Schiffmann R, Kopp JB, Austin HA, 3rd, et al. Enzyme replacement therapy in Fabry disease: a randomized controlled trial. JAMA 2001;285:2743-2749.
8.Choi L, Vernon J, Kopach O, et al. The Fabry disease-associated lipid Lyso-Gb3 enhances voltage-gated calcium currents in sensory neurons and causes pain. Neurosci Lett 2015;594:163-168.
9.Üçeyler N, He L, Schonfeld D, et al. Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy. J Peripher Nerv Syst 2011;16:304-314.
Presenting Author
Julia Grüner
Poster Authors
Julia Grüner
PhD
University Hospital of Wuerzburg
Lead Author
Thomas Klein
Department of Neurology, University Hospital Würzburg, Würzburg, Germany
Lead Author
Maximilian Breyer
Department of Neurology, University Hospital Würzburg, Würzburg, Germany
Lead Author
Nicole Schottmann
University Hospital of Würzburg
Lead Author
Christoph Erbacher
University Hospital Würzburg
Lead Author
Rebecca Mease
Medical Biophysics, Institute for Physiology and Pathophysiology, Heidelberg University, Heidelberg
Lead Author
Topics
- Models: Chronic Pain - Neuropathic