Chapter 6 196 ▲ Figure 3: Localization of Pol III subunits in NES cells. Comparing ratio of nucleus/cytoplasm localization of Pol III subunit A (A) or subunit B (B) in NES of control vs. 4H. Horizontal bars across circles/squares reflect the median. Open circles represent controls, solid circles represent individuals with 4H caused by POLR3A (dark) or POLR3B (light) variants. Data was analyzed using a linear mixed-effects model after removal of influential outliers (indicated with “). * Significance was assessed at p < 0.05, ** P<0.01 or *** P <0.001. Pol III-driven transcript expression unaffected by 4H status To assess potential functional consequences of POLR3 variants we measured abundance of Pol III transcribed products. Specifically, the expression of two tRNA transcripts. Neither tRNAAla and tRNALeu had a significant difference of expression in cell subtypes (Respectively, χ2(2) = 0.570, p = 0.752; Fig. 4A1, and χ2(2) = 3.516, p = 0.172; Fig. 4B1) nor a difference in expression between control and 4H cells (Respectively, χ2(1) = 0.309, p = 0.578; Fig. 4A2, and χ2(1) = 0.086, p = 0.769; Fig. 4B2). Additionally, we investigated expression of Pol III transcript BC200 which was also not significantly different between both cell subtypes and between control and 4H cells (Respectively, χ2(2) = 4.036, p = 0.133; Fig. 4C1; and χ2(1) = 0.174, p = 0.676; Fig. 4C2) ICC values for tRNAAla (16.5%), tRNALeu (0%), and BC200 (32.3%) suggest variable contributions of individual differences to Pol III transcript expression. This indicates that the level of Pol III transcribed transcripts are largely unchanged in 4H cells and minimal inter-individual variability in transcript expression is observed. DISCUSSION In this study, we aimed to identify molecular alterations associated with the neuronal phenotype in 4H leukodystrophy that could point toward primary cell type vulnerabilities. We investigated POLR3 gene expression, Pol III protein levels, and subcellular localization across different cell types. We identified several key alterations, including cell-typedependent POLR3 gene expression and 4H specific reductions in Pol III A and B protein.
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