Fig. 4

Inhibiting NEK6 counteracts p53 pathway activation, DNA damage and axonal transport defects caused by DPRs/C9orf72 mutation. (A) Heat map for p53 pathway-related gene expression from RNA sequencing of DIV80 iCas9 cortical neurons w/wo PR20 treatment. (B) Western blot analysis of DNA damage marker p53BP1 and γH2AX in iCas9-iPSC-derived cortical neurons obtained from iCas9-iPSC-derived cortical neurons transduced with NEK6 sgRNA w/wo PR20 treatment. (C) Western blot analysis for p53-related senescence gene expression (p53 and p21) in all conditions from (B). (D) Representative kymographs of axonal transport recordings in iPSC-derived cortical neurons treated with PR20 peptides and different concentrations of the NEK6 inhibitor, inhibitor 8 (DC12465, DC chemicals, China) starting 3 days before adding PR20. (E) Quantification of total mitochondria, moving mitochondria and ratio of moving mitochondria to total mitochondria normalized to a neurite length of 100 μm during 200 s in (D). (F) Representative kymographs of axonal transport recording in C9orf72 patient and their isogenic control-derived cortical neurons w/wo NEK6 inhibitor 8 treatment (3.5 μM, 3 days). (G) Quantification of total mitochondria, moving mitochondria and ratio of moving mitochondria to total mitochondria normalized to a neurite length of 100 μm during 200 s in (F). (H) LA-PCR of hPRT genes in C9orf72 patient1 and its isogenic control-derived cortical neurons w/wo NEK6 inhibitor 8 treatment. (I) LA-PCR of hPRT genes in C9orf72 patient 2 and its isogenic control-derived cortical neurons w/wo NEK6 inhibitor 8 treatment. (J) Western blot analysis of p53 expression in (I). (K) Western blot analysis of DNA damage marker p53BP1 and γH2AX in neurons from iPSC derived from C9orf72 patient 1 and its isogenic control-derived cortical neurons following NEK6 inhibitor treatment. (L) Western blot analysis of DNA damage marker p53BP1 and γH2AX in neurons from iPSC derived from C9orf72 patient 2 and its isogenic control-derived cortical neurons following NEK6 inhibitor treatment. Data were plotted as mean + SEM. Statistical significance was evaluated with one-way ANOVA and post-hoc Tukey's test; *,**,***, **** P value of <0.05, <0.01, <0.001, <0.0001, respectively