Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy
Fabian Braun, … , Christoph Schell, Tobias B. Huber
Fabian Braun, … , Christoph Schell, Tobias B. Huber
Published April 4, 2023
Citation Information: J Clin Invest. 2023;133(11):e157782. https://doi.org/10.1172/JCI157782.
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Research Article Genetics Nephrology

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Abstract

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Authors

Fabian Braun, Ahmed Abed, Dominik Sellung, Manuel Rogg, Mathias Woidy, Oysten Eikrem, Nicola Wanner, Jessica Gambardella, Sandra D. Laufer, Fabian Haas, Milagros N. Wong, Bernhard Dumoulin, Paula Rischke, Anne Mühlig, Wiebke Sachs, Katharina von Cossel, Kristina Schulz, Nicole Muschol, Sören W. Gersting, Ania C. Muntau, Oliver Kretz, Oliver Hahn, Markus M. Rinschen, Michael Mauer, Tillmann Bork, Florian Grahammer, Wei Liang, Thorsten Eierhoff, Winfried Römer, Arne Hansen, Catherine Meyer-Schwesinger, Guido Iaccarino, Camilla Tøndel, Hans-Peter Marti, Behzad Najafian, Victor G. Puelles, Christoph Schell, Tobias B. Huber

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Figure 1

Podocytes in Fabry disease show persistent lysosomal dysfunction and damage despite enzyme replacement therapy.

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Podocytes in Fabry disease show persistent lysosomal dysfunction and dam...
(A) Transmission electron microscopy (TEM) comparison of foot processes between control and Fabry kidney biopsy before and after ERT with many foot processes widened in Fabry biopsies both untreated and after ERT. Asterisks show Gb3 inclusions in podocytes. Original magnification, ×52,800. (B) Significant decrease of podocyte Gb3 inclusions after ERT but persistence of increased foot process width. (C) Schematic overview of GLA-knockout (KO) podocyte generation by CRISPR/Cas9 genome editing. (D) Western blots show a complete absence of GLA expression in several GLA-KO clones. (E) Abolished GLA activity in 2 KO clones compared with WT cells. (F) Mass spectrometry analysis confirms the accumulation of Gb3-C24-0 isoform in KO cells, normalized upon 96 hours of α-galactosidase therapy (n = 3). (G) TEM shows zebra bodies exclusively in GLA-KO clones (red arrowheads). While WT cells depict a normal ultrastructure, aGAL-treated KO cells have remnant vacuoles (green arrowheads) without zebra bodies. Scale bars: 1 μm. (H) Lysosomal visualization using LAMP1 staining in differentiated WT and KO cells reveals an increased number and size (arrowheads) of lysosomes in the GLA-KO cells. Scale bar: 10 μm. (I) Quantification of lysosomal area (n = 14), pH (n = 12), and ROS production (n = 8). (J) Seahorse XFp experiments confirm normal mitochondrial function in KO cells (n = 8). OCR, oxygen consumption rate. (K) Mitochondrial import receptor subunit (TOM20) staining in WT and KO cells is equally abundant and normally distributed. TEM images confirm a normal mitochondrial ultrastructure in KO cells. Scale bars: 10 μm in immunofluorescence, 500 nm in electron microscopy. Violin plots indicate median (red) and upper and lower quartile (blue). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey’s multiple-comparison test.