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. 2024 Jul 30;111(8):1673–1699. doi: 10.1016/j.ajhg.2024.06.018

Figure 7.

Figure 7

Pathological mechanisms of PAK3 variants resolved using HDF transdifferentiation

(A) Diagram of PAK3 cDNA (GenBank: NM_002578.5) and encoded protein structure. PAK3 nonsense variants are in exons 14 and 16, which encode the kinase domain and are downstream of the auto-inhibitory domain.

(B) PAK3 expression is predominately limited to the brain and pancreas (red) data from The HPA.

(C) PAK3 expression is upregulated during transdifferentiation. Expression of RAC1 family of activated kinases, PAK1, PAK2, and PAK3, during transdifferentiation of HDF to iNeurons. Expression data extracted from srRNA-seq (see Figure 5) and expressed as the mean TPM derived from four replicates per time point.

(D and E) HDFs derived from individuals with the PAK3 variants and transduced with TNA transgene overexpress NERUOG2 and ASCL1 in response to doxycycline treatment for 1, 2, and 5 days. Real-time qPCR assessment of (D) ASCL1 mRNA expression and (E) NEUROG2 mRNA expression. Expression is normalized to ACTB expression.

(F) iNeurons transdifferentiated from HDFs derived from individuals with PAK3 variants display overt neuronal morphology and express neuronal marker genes. Immunofluorescent imaging of day 22 iNeurons: MAP2 (green), TUBB3 (red), PSA-NCAM (blue), SYN1 (red), DAPI (white). Scale bars, 50 μm.

(G) PAK3 variant mRNA expression is reduced in iNeurons. Real-time qPCR of PAK3 expression in day 22 control (n = 3) and nonsense variant iNeurons. Expression normalized to ACTB.

(H) PAK3 variant mRNA is subject to NMD. Real-time qPCR of PAK3 mRNA expression in day 22 control (n = 3) and variant iNeurons treated with or without cycloheximide (CHX) for 24 h prior to RNA collection. mRNA expression is reported as the fold change in expression of CHX treated versus non-CHX treated cells. Expression normalized to ACTB.

(I) PAK3 variant mRNA is lowly expressed in transactivated HDFs. PAK3 was transactivated in HDFs derived from n = 3 control individuals and individuals with PAK3 variants. Isolated RNA was subjected to real-time qPCR. Expression is normalized to ACTB.

(J) PAK3 variant mRNA acquired through transactivation is subject to NMD. Real-time qPCR of PAK3 expression transactivated HDFs treated with or without cycloheximide (CHX) for 24 h prior to collection. Expression is reported as the fold change in expression of CHX-treated versus non-CHX-treated cells. Expression normalized to ACTB. Statistical analysis was determined by ordinary one-way ANOVA with Tukey’s multiple comparison test. Significance set as ∗p < 0.05, ∗∗ <0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. All error bars respresent standard deviation.