Rbfox Splicing Factors Promote Neuronal Maturation and Axon Initial Segment Assembly

Abstract

Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.

Keywords: AnkG; Rbfox; actin cytoskeleton; alternative splicing; axon initial segment; neuronal maturation.

Figure 1. In vitro maturing ventral spinal neurons derived from ESCs undergo developmental splicing changes paralleling those observed in mouse cortex

(A) Map2 staining shows morphological changes of motor neurons from day 2 (D2) to day 15 (D15) of in vitro culture after plating. Scale bar: 50 μm. (B) A representative trace from whole-cell patch-clamp recording of maturing motor neuron on day 5 after current injection. (C) Maturation of ventral spinal neurons is staged by their gene expression (top) or splicing (bottom) profiles using cortex samples as a reference. Transition of maturation stages for motor neurons on day 1, day 5 and day 10 are indicated. Additonal samples derived from different types of neurons or neuronal tissues are also included for comparison. (D) Four modules of exons show distinct temporal patterns of splicing in developing cortex (left) and spinal neurons (right)(Weyn-Vanhentenryck et al, 2018). These exons were identified and ordered based on their developmental splicing changes in the cortex and their splicing profiles in spinal neurons are shown in the same order. Exons in each module are further divided into two groups (e.g., M1+ and M1-) based on the direction of splicing changes E) Overlap between exons showing developmental splicing changes in the cortex and exons showing splicing changes in developing spinal neurons. Exons with increased or decreased inclusion in each module and direction are shown separately. The number of exons in each group is indicated.

Figure 2. Depletion of Rbfox proteins in ventral spinal neurons perturbs the global splicing program with only minor changes at the steady-state mRNA level

(A) Schematic illustration of the Rbfox triple knockout (tKO) mouse ES cell line generated by CRISPR/Cas9-mediated genome engineering. Each Rbfox gene is disrupted by an 11-nt insertion that includes in-frame stop codons and a restriction enzyme (RE) digestion site for genotyping. The triple knockout was confirmed by PCR genotyping of ESCs using primers flanking the mutated site and restriction digestion. Depletion of Rbfox proteins was confirmed by immunoblot analysis in in vitro differentiated neurons; GAPDH is a loading control. (B) Comparison of motor neuron differentiation efficiency from day 2 WT and Rbfox tKO motor neuron culture. Representative immunostaining images are shown on the left. Map2 and Hb9 are pan-neuronal and motor neuron markers, respectively. Scale bar: 50 μm. The result of quantification is shown on the right (two-way ANOVA with post hoc Bonferroni’s multiple test correction, n.s., p>0.05, n=4); error bars represent standard error of the mean (S.E.M.). (C) Differential gene expression analysis of WT versus Rbfox tKO neurons. Genes with significant changes on day 10 are shown in blue (fold change >1.5, FDR<0.05). Rbfox1, 2 and 3 genes are highlighted. (D) Differential splicing analysis of annotated cassette exons in WT versus Rbfox tKO neurons. The scatter plot shows the magnitude of splicing changes (ΔΨ) at the two time points. Only exons with significant splicing changes (|ΔΨ|≥0.1 and FDR≤0.05) on day 5 or day 10 are shown. The correlation of splicing changes between the two time points is indicated. (E) The total number of differentially spliced AS events in WT versus Rbfox tKO neurons (|ΔΨ|≥0.1 and FDR≤0.05). Novel AS events were identified in the mouse cortex transcriptome. CASS, cassette exon; TACA, tandem cassette exons; MUTX, mutually exclusive exons; ALT5, alternative 5′ splice sites; ALT3, alternative 3′ splice sites. (F) Two examples of Rbfox-regulated alternative exons. RNA-seq, Rbfox1–3 pooled CLIP data and the Rbfox binding (U)GCAUG motif sites are shown as separate tracks in each panel. (G) Integrative modeling defines direct Rbfox targets. Schematic of the integrative modeling framework is shown at the top. The comparison of Rbfox targets defined using motor neuron culture data and those defined using data derived from mouse brain and other sources (without including motor neuron data) is shown at the bottom. See also Figures S1–S3.

Figure 3. Rbfox tKO neurons retain an embryonic-like splicing profile and immature electrophysiological properties

(A, B) Comparison of maturation stages of WT and Rbfox tKO neurons based on splicing profiles (A) and gene expression profiles (B). (C) Maturation of WT and Rbfox tKO neurons is staged using splicing profiles of Rbfox targets. Results from target exons of several other RBPs (Nova, Mbnl and Ptbp) are shown for comparison. (D) Representative gene ontology (GO) terms enriched in Rbfox target transcripts defined by Bayesian network analysis. See also Table S5 for the complete list. (E) Whole-cell patch-clamp recording of maturing motor neuron on day 5. Representative traces of action potential firing in WT and tKO motor neurons in response to current injection are shown. (F) Quantification of whole-cell patch-clamp measurements. For each parameter, the direction of change during neuronal maturation is indicated by the arrow on the right of the bar plot (** p<0.01, * p<0.05, t-test, N=34 in each group). Error bars represent S.E.M. See also Figure S4.

Figure 4. Rbfox-regulated exons are enriched in genes encoding regulators and components of the AIS

(A) Genes encoding defined AIS components are shown in the schematic diagram. Genes with Rbfox-regulated exons as defined by Bayesian network and RNA-seq are indicated in red. Additional AIS genes reported in the literature are also shown on the right. (B) Magnitude of Rbfox-dependent splicing changes in AIS genes as measured by RNA-seq and RT-PCR validation. Two deregulated alternative exons of Scn8a and Ank3 are shown in the scatter plot separately. Exons with asterisks in the scatter plot correspond to those tested by RT-PCR.

Figure 5. Rbfox tKO motor neurons show severe pertubation of AIS and AnkG organization

(A) Immunostaining analysis showing AIS morphology in day 5 WT and Rbfox tKO motor neurons. Motor neurons are identified by Hb9 staining. The arrowheads mark the beginning and the end of the AIS. Scale bar: 50μm. (B) Quantification of WT and Rbfox tKO motor neurons that lack AnkG acumulation in the proximal axon (left) or have reduced AIS length based on AnkG staining (right) during maturation (two-way repeated measures ANOVA, post hoc Bonferroni’s multiple comparisons test; ****p<0.0001, ***p<0.001, **p<0.01, n.s. p>0.05; N ranges from 45 to 91 for each group). Error bars represent S.E.M. (C, D) Immunostaining of axonal marker Tau-1 (C) and βIV-spectrin (D) on day 5 WT and tKO motor neurons. Scale bar: 50 μm. (E) Representative 3D-STORM images and quantitative analysis of AnkG in the AIS in day 10 WT and tKO neurons. Left: 3D-STORM images of immunolabeled AnkG in AIS. tKO results are classified into sparse, patchy and periodic patterns. Alternation of low and high AnkG density regions in the patchy pattern is indicated by the white ovals and magenta arrowheads, respectively. The color scale used to indicate depth in z is shown at the bottom. Scale bars: 1 μm. Middle and right: Fourier transform (middle) and autocorrelation analyses (right) of AnkG distribution in the indicated regions of AIS in the left panel (grey dashed lines). (F) Box plot of average autocorrelation amplitude of AnkG in all WT and tKO AIS (p=0.005, Wilcoxon-Mann-Whitney test, N=13 AIS for WT and N=23 for tKO neurons). The red line indicates the threshold used to classify periodic vs. non-periodic AIS. (G) Stacked column graph showing fractions of AnkG patterns out of all analysed AIS. See also Figures S5 and S6.

Figure 6. Rescue of AIS defects in Rbfox tKO neurons by overexpression of individual Rbfox protein

(A) Subcelullar localization of the exogenously expressed Rbfox proteins in motor neurons. Representative immunostaining images are shown on the left (scale bar: 10 μm) and quantification of nuclear protein concentration based on fluorescence intensity is shown on the right (***p<0.001, t-test, N ranges from 60 to 63 for each group). (B) Immunoblot analysis to validate expression of FLAG-tagged Rbfox proteins after plasmid transfection (top). Rescue of splicing of two representative Rbfox target exons is also shown at the bottom. (C) Representative images for AIS analysis upon overexpression of Rbfox1, Rbfox2 and Rbfox3 in Rbfox tKO motor neurons, as well as WT and tKO control. Scale bar: 50 μm. (D) AIS quantification in control WT and Rbfox tKO motor neurons, and Rbfox tKO motor neurons upon overexpression of individual FLAG-tagged Rbfox protein (one-way ANOVA with post hoc Tukey’s multiple test correction; ****p<0.0001, ***p<0.001, **p<0.01; N ranges from 60 to 63 for each group). Error bars represent S.E.M. See also Figure S7.

Figure 7. Inclusion of a developmentally regulated alternative exon upstream of AnkG ZU5-1 domain negatively affects interaction of AnkG with βIV- and βII-spectrins

(A) Top: A schematic illustration of AnkG protein domains. Arrowheads indicate five alternative exons differentially spliced in Rbfox tKO neurons on day 5 or day 10. The candidate 33-nt cassette exon immediately upstream of the ZU5-1 domain is highlighted in red. Bottom: Structural modelling of the ZU5-1/β-spectrin complex. A published structural model of βI-spectrin repeats 13–15 (gray) and AnkR ZU5-1 (blue) domain (PDB ID: 3KBT) is shown on the left and structural prediction of AnkG ZU5-1 (blue) including the segment encoded by the candidate alternative exon (red) is shown on the right. The three crucial interaction interface residues (two arginines at the bottom and one alanine on the right) are highlighted in orange in both models. (B) RNA-seq quantification of the candidate cassette exon in Ank3 (top) and its homolog in Ank2 (bottom) in WT and Rbfox tKO neurons on day 5 and day 10 (left) and in developing mouse cortex (right). (C) CLIP tags indicating positions of Rbfox, Mbnl2 and Nova binding sites in Ank3 (top) and Ank2 (bottom) in the alternatively spliced region are shown and the strongest peaks are indicated by blue and red arrowheads, depending on their positions relative to the alternative exon. (D) Shematics of constructs for co-immunoprecipitation of AnkG/AnkB ZU5–1 domains with βII-/βIV-spectrin repeats 13–15 overexpressed in NIH/3T3 cells. The peptide encoded by the candidate alternative exon in AnkG and its homolog in AnkB is also shown. (E, F) Immunoblot analysis of the co-immunoprecipitation experiments. The + and – signs denote the combination of the transfected plasmid constructs. In all experiments, anti-FLAG antibodies were used for immunoprecipitation. (E) Comparison of AnkG inclusion and exclusion isoforms for β-spectrin binding. (F) Comparison of AnkG and AnkB exclusion isoforms for β-spectrin binding. (G) Immunostaining analysis showing AIS in WT motor neurons transfected with AnkG ZU5in-3×HA or AnkG ZU5ex-3×HA plasmid vector. AIS is stained using AnkG antibody that detects only the endogenous protein, and the overexpressed ZU5-1 domain isoforms are detected by anti-HA antibody. The arrowheads mark the beginning and the end of the AIS. The arrow indicates an HA-positive motor neuron showing weak and distributed AnkG staining. (H) Quantification of AIS presence and length based on AnkG staining (one-way ANOVA with post hoc Tukey’s multiple test correction; ****p<0.0001, ***p<0.001, **p<0.01, n.s. p>0.05, N ranges from 47 to 70 for each group). Error bars represent S.E.M. Scale bar: 50 μm.

Figure 8. Deregulation of the alternative exon upstream of AnkG ZU5-1 domain negatively affects AIS assembly

(A) A schematic shows generation of ESC lines for constitutive expression of the AnkG exclusion (AnkGdel) or AnkG inclusion (AnkGins) isoform by deletion and subsequent re-insertion of the candidate alternative exon in the Ank3 (AnkG) gene. (B) RT-PCR analysis of the candidate exon inclusion in WT, AnkGdel and AnkGins maturing neurons. (C) Immunostaining analysis showing AIS in day 5 WT, AnkGdel and AnkGins motor neurons. The red arrowheads mark the beginning and the end of AIS. The arrow shows an axon with weak and distributed AnkG staining. Scale bar: 50 μm. (D) Map2 restriction in somatodendritic compartment is perturbed in AnkGins motor neurons without AIS. Zoom-in views of the boxed area in panel (C) are shown at the top and respective axonal tracing fluorescence intensity profiles for Map2 and AnkG are at the bottom. The red arrows indicate AnkG puncta in the axon. (E) Quantification of AIS presence and length in WT, AnkGdel and AnkGins motor neurons on day 5 and day 10 based on AnkG staining (two-way ANOVA with post hoc Tukey’s multiple test correction; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05; N equals 66 for both groups). Error bars represent S.E.M. (F) Quantification of significantly changed electrophysiological characteristics from whole-cell patch-clamp measurements of WT and AnkGins motor neurons on day 5. (t-test, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05; N ranges from 25 to 28 per group). Error bars represent S.E.M. (G) Correlation of action potential firing and AIS length in WT and AnkGins motor neurons. Post hoc quantification of AIS was performed in motor neurons based on AnkG staining after whole-cell patch-clamp recording. The gray and the blue datapoints represent individual WT and AnkGins motor neurons, respectively with the dotted lines indicating the average values. (H) Immunostaining and whole-cell patch clamp recordings showing the maximum number of fired action potentials after current injection for representative motor neurons, denoted i, ii and iii in panel (G). See also Figures S9 and S10. See also Figure S8.