Page 45 - Read Online

Ryan et al. J Transl Genet Genom. 2025;9:48-61 https://dx.doi.org/10.20517/jtgg.2024.87 Page 52

RESULTS
Lysosomal morphology in FD podocytes
TEM analysis revealed distinct morphological differences in lysosomes between control and FD podocytes
at day 10 of differentiation. Control podocytes displayed well-formed, rounded lysosomes that appeared
small and electron-dense, uniformly distributed within the cytoplasm [Figure 1A]. In contrast, FD
podocytes exhibited markedly enlarged and irregularly shaped lysosomes, presenting as electron-dense
deposits [Figure 1B] or enlarged myelin bodies [Figure 1C], both characteristic of FD. These lysosomal
inclusions were suggestive of the accumulation of undigested substrates, consistent with the pathological
features observed in FD.

Quantitative analysis of the lysosomal area demonstrated a significant increase in lysosomal area in FD
podocytes compared to controls [Figure 1D and E]. These findings highlight lysosomal enlargement and
morphological abnormalities present in FD podocytes, correlating with the known dysfunction of α-Gal A
activity and Gb3 substrate accumulation characteristic of the disease.

Proteomic analysis shows clear differences between FD and control podocytes
iPSC-derived podocytes from a FD patient carrying the p.Met284Thr GLA variant were previously
confirmed to have a significant reduction in α-Gal A activity and increased Gb3 accumulation compared to
control podocytes . In Figure 2A, Principal Component Analysis (PCA) was used to dimensionally
[10]
condense the large proteomic data set by reducing it to linear principal components that capture the
differences between FD and control podocytes. PC1 represents 29.4% of variance along the X-axis, while
PC2 represents 11.2% of variance along the Y-axis. The results showed that FD podocytes grouped distinctly
from controls, highlighting major differences in their protein profiles.

Hierarchical clustering in the heatmap [Figure 2B] showed distinct patterns of protein abundance,
represented by clear clusters of proteins that were either higher or lower in FD podocytes compared to
controls. This highlights specific molecular changes, including pathways involved in lysosomal function and
cellular organization. Figure 2C provides a Venn diagram that shows the overlap and distinct proteomic
profiles between control and FD podocytes. A total of 5,971 proteins (89%) were shared between the control
and FD podocytes, indicating a significant overlap. However, 581 proteins (9%) were uniquely identified in
control podocytes, with 188 proteins (3%) unique to FD podocytes. This indicates that although there is a
considerable shared proteomic baseline, FD podocytes exhibit a distinct subset of proteins, highlighting the
specific molecular alterations associated with the pathogenic GLA variant and the resulting lysosomal
dysfunction.

Further analysis of key enzymes involved in glycosphingolipid metabolism provided deeper insights into the
specific pathways disrupted in FD podocytes. Box plots for GLA, glucosylceramindase (GBA), and
galactosylceramidase (GALC) proteins [Figure 2D] showed significant alterations in their expression levels
in FD podocytes compared to controls. GLA (encoding α-Gal A) was expectedly reduced in FD podocytes,
while GBA and GALC, both enzymes involved in glycosphingolipid degradation, displayed compensatory
upregulation. These data suggest potential cellular responses to the accumulation of Gb3 and other
substrates in FD podocytes. A summary of the glycosylceramide catabolic process is represented in
Figure 2E, indicating how protein abundance shifts observed in FD podocytes align with known metabolic
disruptions. The decreased activity of α-Gal A leads to Gb3 accumulation, while compensatory upregulation
of GBA and GALC suggests an attempt to process excess glycolipids through alternate degradation
pathways.