Fig. 5: Modeling of THOC6 variant pathogenesis in human cerebral organoids.

A Cerebral organoid differentiation protocol. ND, neural differentiation. B Immunostaining of PH3, N-Cadherin, apoptosis marker cleaved caspase−3 (C.CASP3), and Hoechst in day 28 human cerebral organoids differentiated from unaffected and affected iPSCs, highlighting differences in neural rosette (NR) morphology. X40 magnification; Scale bar: 50 μm. UNAFF, unaffected; AFF, affected. Representative images for UNAFF and AFF are THOC6^E188K/+ and THOC6^E188K/E188K, respectively. C Quantifications of NR thickness (left), NR area (middle), Hoechst+ cells per NR. UNAFF, unaffected (black; quantifications from THOC6^E188K/E188K and THOC6^W100*/W100*); AFF, affected (red; quantifications from THOC6^E188K/+ and THOC6^W100*/+). NR (organoid) biological replicates analyzed across three differentiation replicates per genotype: unaffected, n = 67 (15) biological replicates; affected, n = 34 (10) biological replicates. Data shown as mean ± SEM. Significance, two-tailed unpaired t test; p = <0.0001 (thickness), p = <0.0001 (area), p = <0.0001 (Hoescht + ). D Fraction of C.CASP3+ cells per NR for THOC6^W100*/+ and THOC6^E188K/+ controls and THOC6^W100*/W100* and THOC6^E188K/E188K affected organoids. UNAFF, unaffected (black; quantifications from THOC6^E188K/E188K and THOC6^W100*/W100*); AFF, affected (red; quantifications from THOC6^E188K/+ and THOC6^W100*/+). NR (organoid) biological replicates analyzed across three differentiation replicates per genotype: unaffected, n = 67 (15) biological replicates; affected, n = 34 (10) biological replicates^. Data shown as mean ± SEM. Significance, two-tailed unpaired t test, p = <0.0001. E Immunostaining of EDU, KI67, DCX to assess timing of differentiation in day 28 organoids. Representative images for UNAFF and AFF are THOC6^E188K/+ and THOC6^E188K/E188K, respectively. F Quantifications for E; UNAFF, unaffected (black; quantifications from THOC6^E188K/E188K and THOC6^W100*/W100*); AFF, affected (red; quantifications from THOC6^E188K/+ and THOC6^W100*/+). NR (organoid) biological replicates analyzed across three differentiation replicates per genotype: unaffected, n = 187 (87) biological replicates; affected, n = 157 (67) biological replicates. Data shown as mean ± SEM. Significance, two-tailed unpaired t test; p = <0.0001 (KI67 + /Hoescht + ), p = 0.0121 (KI67+EdU + / EdU + ), p = 0.0008 (DCX+EdU + /EdU + ). G Growth rate of organoids across genotypes measured by cross section area (μm) from days 21-42. Genotypes: THOC6^+/+ (black), THOC6^E188K/+ (dark purple), THOC6^E188K/E188K (purple), THOC6^W100*/+ (dark green), THOC6^W100*/W100* (green). H Schematic of proposed model of THOC6 pathogenesis, depicting disruption of RNA processing in human neural cells leading to impaired proliferation and differentiation timing during corticogenesis. Additionally, given ALYREF’s defined role in multimerizing several TREX complexes coating mRNPs³³, impaired recruitment of ALYREF to TREX complexes has implications for mRNP compaction in THOC6 LOF cells.