Background & Aims
Neuropathic pain affects at least 1-2 % of the population.1 Partly, neuropathy is tied to genetic mutations, utilizable to derive cellular models for insights in disease specific and general pain processes. Fabry disease (FD) affects multiple organs including dorsal root ganglia (DRG), leading to acral and triggerable pain.2 Sensory neurons (SN) residing in DRG are mediators of somatosensory and nociceptive sensation and are altered on transcriptome and functional level in animal models.3, 4 Mutations in the GLA gene result in accumulations of globotriaosylceramide (Gb3), yet cellular mechanisms are incompletely understood. Enzyme therapy is insufficient in alleviating pain5 and inaccessibility of human SN represents a major bottleneck for better understanding and effective treatment. We mapped pathologic pathways in an isogenic human induced pluripotent stem cell derived (iPSC) sensory neuron (iSN) line with a c.1091_1093del (p.S364del, hemizygous) mutation versus its healthy control.
Methods
Both characterized iPSC lines were differentiated into iSN via an established protocol.6 After 10 days of differentiation, iSN were split into 6-wells with base matrigel coating and matured for 6 weeks. We compared the healthy (CTR) and Fabry line (ISO-FD), along the ISO-FD line with enzyme supplementation (ISO-FD-AGAL), consisting of 1.32 ?g/ml ?-galactosidase A (AGAL; Shire Human Genetic Therapies Inc, Lexington, MA, USA) added biweekly to mimic enzyme replacement therapy. Neurons were mechanically detached with cold PBS and after centrifugation lysed in RIPA buffer containing protease inhibitor. Whole protein was determined via BCA assay. After in-gel digestion with trypsin, the proteome was assessed via liquid chromatography tandem mass spectrometry. Deregulated proteins were subjected to pathway analysis via DAVID and metascape.
Results
We detected 4190 proteins in total with 275 dysregulated proteins in ISO-FD versus CTR. Further, comparing ISO-FD with AGAL treatment against CTR revealed 156 dysregulated proteins. Pathway analysis via DAVID indicated enrichment of Golgi processes (enrichment score 3.56; with GO_CC golgi apparatus padj = 0.002, GO_CC golgi membrane padj = 0.018, and KW Golgi apparatus padj = 0.012) and mitochondria (enrichment score 2.66; GO_CC mitochondrion padj = 0.001; KW mitochondrion padj = 0.003, and GO_CC mitochondrion inner membrane padj = 0.018) as most relevant clusters in FD-ISO. Protein-protein interaction analysis within metascape indicated Golgi-associated vesicle biogenesis (p_Log10 = -11.3) and mitochondrial translation (p_Log10 = -12.8) as two key pathways altered in iSN of FD-ISO. The associated proteins were almost exclusively upregulated compared to CTR iSN. Modified pathways between FD-ISO and FD-ISO-AGAL were largely overlapping, yet less enriched in the AGAL treated iSN.
Conclusions
We provide novel insights in the cellular mechanisms underlying FD in human sensory neurons. Gb3 accumulation may provoke increased Golgi-mediated vesicle and lysosome production and perturbation in membrane trafficking. Thus, transport of receptors and ion channels to or internalization from the membrane, essential for homeostatic excitability, may be altered. Elevated mitochondrial translation may indicate a higher energy demand or faster turnaround time of mitochondria in the FD iSN model, converging with morphologically alterations previously observed in FD cells.7, 8 AGAL enzyme incubation partially attenuated proteomic changes, but was not sufficient to fully restore Golgi and mitochondrial protein overexpression within 6 weeks of supplementation.
References
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2.Miller JJ, Kanack AJ, Dahms NM. Progress in the understanding and treatment of Fabry disease. Biochim Biophys Acta Gen Subj. 2020 Jan;1864(1):129437.
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4.Kummer KK, Kalpachidou T, Kress M, Langeslag M. Signatures of Altered Gene Expression in Dorsal Root Ganglia of a Fabry Disease Mouse Model. Front Mol Neurosci. 2017;10:449.
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6.Klein T, Gruener J, Breyer M, Schlegel J, Schottmann NM, Hofmann L, Gauss K, Mease R, Erbacher C, Finke L. Small fibre neuropathy in Fabry disease: A human-derived neuronal in vitro disease model. bioRxiv. 2023:2023.08. 09.552621.
7.Klug K, Spitzel M, Hans C, Klein A, Schottmann NM, Erbacher C, Üçeyler N. Endothelial Cell Dysfunction and Hypoxia as Potential Mediators of Pain in Fabry Disease: A Human-Murine Translational Approach. Int J Mol Sci. 2023 Oct 21;24(20).
8.Schumann A, Schaller K, Belche V, Cybulla M, Grünert SC, Moers N, Sass JO, Kaech A, Hannibal L, Spiekerkoetter U. Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells. J. Inherit. Metab. Dis. 2021;44(4):1039-50.
Presenting Author
Christoph Erbacher
Poster Authors
Christoph Erbacher
PhD
University Hospital Würzburg
Lead Author
Aneeta Andrews
Department of Neurology, University Hospital Würzburg, Würzburg, Germany
Lead Author
Maximilian Breyer
Department of Neurology, University Hospital Würzburg, Würzburg, Germany
Lead Author
Stephanie Lamer
Rudolf-Virchow-Zentrum, University Würzburg, Würzburg, Germany
Lead Author
Till Sauerwein
PhD
ZB MED – Information Centre for Life Sciences, Cologne, Germany
Lead Author
Konrad Förstner
Prof.
ZB MED – Information Centre for Life Sciences, Cologne, Germany
Lead Author
Andreas Schlosser
PhD
Rudolf-Virchow-Zentrum, University Würzburg, Würzburg, Germany
Lead Author
Nurcan Üçeyler
MD
Department of Neurology, University of Würzburg, Germany
Lead Author
Topics
- Models: Chronic Pain - Neuropathic