Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons

Abstract

We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.

Keywords: ACTL6B; genetic engineering; intellectual disability; neurodevelopment; seizure; stem cells.

Figure 1

Location of Mutations in ACTL6B Found in Individuals with Potential Recessive or Dominant Disease-Causing Mutations (A) Photos of individuals with ACTL6B mutations. Note broad mouth of individuals D1, D2, D3, and D7, diastema in D1, D3, D7, bulbous tip of the nose in all D individuals, and hypertelorism with telecanthi in individual D8. Lower right: MRI images of individuals with recessive ACTL6B mutations. For individual R4, note white matter T2 hyperintensity (arrows). For individual R8, note enlarged lateral ventricles and asymmetric gyral pattern (left, arrows). On the right, note thin corpus callosum (arrows). (B) Linear graph of mutations in ACTL6B (introns not drawn to scale). (C) Conservation of the residues affected by amino acid substitutions. (D) 3D model generated with SWISSMODEL based on S. cerevisiae Arp4 (yeast homolog of ACTL6B), visualized with Swiss-PdbViewer showing that recessive mutations are not focused in one region. Note, however, that the dominant mutations seem to lie at the periphery of the protein and thus they might affect protein-protein interactions.

Figure 2

Generation of iPSC-Derived Neurons for BAF53 Studies (A) Representative images of quality control staining done on iPSCs. (B) Representative quality control staining on NPC cultures. (C) Representative staining of control cells for TUJ1 and MAP2 at D15 of differentiation. (D) Representative trace of miniature EPSCs from D25 neurons held at −40 mv. (E) Representative recordings showing spontaneous activity of D25 neurons in current-clamp mode. (F) Trace of a hyperpolarizing pulse showing a depolarizing sag followed by multiple rebound action potentials. The first action potential is shown at a higher temporal resolution. All scale bars represent 40 μm.

Figure 3

Comparison of Control and ACTL6Bext^∗33 before and after Expression of ACTL6B (A) Diagram illustrating the production of control and ACTL6Bext^∗33 iPSC-derived NPCs from fibroblasts. (B) ACTL6B expression normalized to GAPDH expression plotted against number of days of differentiation of NPCs. n = 4, error bars represent standard error around the mean. (C) Expression of key genes in the SWI/SWF complex in 706 ACTL6Bext^∗33 and control NPCs in proliferating and post-mitotic states. Genes are normalized to GAPDH expression. n ≥ 3; Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01. (D) Western blots assessing the level of proteins encoded by the genes displayed in (C).

Figure 4

Generation and Characterization of ACTL6B KO Neurons Reveals a Loss of Dendrites (A) Diagram of the experimental approach taken to generate ACTL6B KO NPCs. (B) Sanger sequencing traces of two ACTL6B KO lines. (C) ACTL6B expression in control and ACTL6B KO NPCs at a D0 and D5 time point (n ≥ 3). (D) Western blots assessing the protein levels of BAF53A/B in ACTL6B KO lines. (E) Representative TUJ1 and MAP2 staining of control and ACTL6B KO D15 immature forebrain neurons. (F) Quantification of the surface area of the nucleus in the cell lines shown in (E) (n > 50). Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01.

Figure 5

Repair of the ACTL6B c.1279del Mutation Restores Morphological and Dendritic Deficits (A) Schematic detailing ACTL6B CRISPR repair. (B) Sanger sequencing traces of a successful repair (SR) and unrepaired (UR) cell line generated from ACTL6Bext^∗33 cell line. (C) ACTL6B expression in SR and UR NPCs at a D5 time point (n = 6). (D) Representative TUJ1 and MAP2 staining taken from SR and UR forebrain immature neurons at D15. Scale bars represent 40 μm. (E) Quantification of the surface area of the nucleus and soma and the length of projections in the cell lines shown in (D) (n > 50). Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

ACTL6Bext^∗33 Variant Leads to Increased Binding of BRG1-BAF Complex to the Genome (A) Diagram illustrating the ChIP-seq experiment. (B) Venn diagram showing overlap of genes that the BRG1 complex is bound to. (C) Decreased binding at all 382 FDR significant sites in control cells compared to ACTL6Bext^∗33 cells (pink dots are significant, while blue dots are not). (D) Proportion of BRG1 binding sites found in relation to their proximity to a gene. (E) Gene ontology analysis of differentially bound regions. (F) Within-subjects differential binding across developmental stages (D0 and D5) showing decreased binding in D0 compared to D5 in ACTL6Bext^∗33 cells (pink dots are significant, while blue dots are not). Genes showing a significant difference (FDR-adjusted p values [Benjamini-Hochberg] ≤ 0.05) in D5 relative to D0 using a GLM as implemented in DESeq2.

Figure 7

External Validity in Multiple ACTL6B Mutant Models in Human Neurons (A) TPPP and FSCN1 expression in initial RNA-seq (n ≥ 4) and qPCR (n ≥ 3) data (ACTL6Bext^∗33 versus control); unrepaired (UR) ACTL6Bext^∗33 versus ACTL6Bext^∗33 successful repair (SR) (n = 6); and ACTL6B KO versus isogenic control cells (n = 5). Results are represented as mean ± SEM. Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (B) Experimental plan for generation of multiple human neuronal cell lines expressing various mutant ACTL6B constructs. (C) Brightfield and GFP images demonstrating high transfection of ACTL6B constructs. (D) mRNA expression in transfected ACTL6B KO NPCs at D5 time points of ACLT6B, TPPP, and FSCN1 (n = 3). Results are represented as mean ± SEM. Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 8

Neurons Derived from an Individual with a Compound Mutation in ACTL6B Show a Similar Phenotype to ACTL6Bext33 and ACTL6B KO Neurons (A) Schematic showing generation of ACTL6B compound mutant NPCs. (B) Sanger sequencing traces of ACTL6B compound mutant and control cell line at both identified point mutations in ACTL6B. (C) Representative TUJ1 and MAP2 staining of control and ACTL6B compound mutation immature forebrain neurons. (D) Quantification of the surface area of the nucleus in the cell lines shown in (E). (E) TPPP and FSCN1 expression in ACTL6B compound mutant versus control cells at mitotic (D0) and post-mitotic (D5) time points (n > 50). Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01.