Figure 7. Target deconvolution using unbiased quantitative proteomics in AP4B1^KO SH-SY5Y cells and AP-4-HSP patient-derived iPSC-neurons treated with C-01.

(a – c) Differential protein enrichment analysis. Statistical testing was done using protein-wise linear models and empirical Bayes statistics. Proteins were considered as differentially enriched with a false discovery rate of < 0.05 and a log₂ fold change > 0.3. (a) SH-SY5Y cells: 8141 unique proteins were analyzed. PCA of the top 500 variable proteins shows robust separation between experimental conditions. The volcano plot summarizes differential protein enrichment for AP4B1^WT and AP4B1^KO cells pooled into two groups, vehicle vs. C-01 treated. Differentially enriched proteins are depicted in black. Proteins with the most consistent enrichment profiles across all experimental conditions (see Supplementary Fig. 6a-d) are colored and labeled in red. (b) iPSC-derived neurons: 7386 unique proteins were analyzed. PCA of the top 500 variable proteins shows robust separation between experimental conditions. The volcano plot summarizes differential protein enrichment for control and patient-derived neurons pooled into two groups, vehicle vs. C-01 treated. Differentially enriched proteins are depicted in black. Proteins with the most consistent enrichment profiles across all experimental conditions (see Supplementary Fig. 6e-h) are colored and labeled in red. (c) Integrated analysis of SH-SY5Y cells and iPSC-derived neurons: 5357 unique proteins were analyzed. The volcano plot summarizes differential protein enrichment for control and AP-4-deficient cells pooled into two groups, vehicle vs. C-01. Proteins with the most consistent enrichment profiles across all experimental conditions (see Supplementary Figure 6i-l) are colored and labeled in red. The dot plot summarizes dysregulated Reactome pathways of the pooled analysis. Pathways were considered differentially expressed with an FDR < 0.05. (d) The RAB protein family members RAB1B, RAB3C and RAB12 showed the most consistent profiles in response to C-01 treatment and were selected for further analysis. LFQ intensities in SH-SY5Y cells (AP4B1^WT and AP4B1^KO pooled) and neurons (control and patient pooled) are shown. Statistical testing was done using pairwise T-tests. P-values were adjusted for multiple testing using the Benjamini-Hochberg procedure. (e) LFQ intensities of RAB3C and RAB12 in AP4B1^WT (n = 11 samples) and AP4B1^KO (n = 10 samples) SH-SY5Y cells, as well as control (n = 6 samples) and patient-derived (n = 6 samples) iPSC-derived neurons show a high degree of correlation measured by the Pearson correlation coefficient (r). While there was no difference between genotypes (not shown), C-01 treated cells showed reduced protein levels of both RAB3C and RAB12.