Figures and data
A Human iPSC-based model of TDP43 proteinopathy in MNs
(A) Schematic depicting genome editing of healthy iPSCs to knock-in GFP into the C-terminus of endogenous TARDBP, encoding TDP43. Yellow rectangle indicates the TDP43 last exon. Red vertical line indicates the STOP codon. Blue rectangle indicates the TDP43 3’UTR. The resulting TDP43-GFP iPSCs are differentiated into motor neurons (MNs) and transduced with AAVs encoding anti-GFP nanobodies. The “NES” nanobody includes a sequence for a strong nuclear export signal, which transports nuclear TDP43-GFP into the cytoplasm. The control nanobody (lacking NES) has no effect on TDP43-GFP localization.
(B) Representative images depicting TDP43 localization in two homozygous TDP43-GFP lines (E8, E5). MNs were transduced at day 20 of the differentiation process and fixed at day 40 for the immunostaining. TDP43 is expressed in the nucleus in the presence of the control nanobody and relocates to the cytoplasm in the presence of the NES nanobody. TDP43-GFP indicates signal from the anti-GFP antibody while TDP43 indicates signal from the anti-TDP43 antibody. Neurons were also stained with neurofilament-M (red) and Hoechst 33342 (blue nuclear stain). Scale bar = 50 µm.
(C) Quantification of the % of nuclear TDP43 in individual neurons transduced with control or NES nanobodies.
(D) Quantification of morphological defects in NES nanobody treated MNs versus control. Mislocalized TDP43 (NES) causes a reduction in dendritic tree length (Dendrites) and soma swelling (Soma), compared to neurons expressing nuclear TDP43 (control).
(E) Quantification of cleaved caspase-3 (CC3) expression as a marker of apoptosis. Cytoplasmic TDP43 causes significant elevation in apoptosis compared to nuclear TDP43. Panels C, D, and E display data for the two homozygous TDP43-GFP lines (E8 and E5), transduced with nanobodies at day 20 and fixed for staining at day 40. N = 3 independent differentiations per clone. At least 100 neurons were included per condition per replicate.
(F) Representative images showing cytoplasmic aggregation of TDP43 at day 40 in homozygous TDP43-GFP MNs transduced with the NES nanobody at day 18. Scale bar = 15 µm.
(G) Upper panel: representative western blot image of soluble TDP43-GFP in NES versus control MNs at day 42, 22 days post-transduction with AAV nanobodies. Lower panel: Quantification of soluble TDP43-GFP from western blot. N = 3 independent differentiations, two for E5 iPSCs, one for the E8 iPSCs.
*** indicates p < 0.001.
Transcriptional Consequences of TDP43 Mislocalization
(A) Principal component analysis of gene counts for E8 and E5 iPSC-MNs.
(B) Volcano plot displaying genes differentially expressed due to TDP43 mislocalization. Red: upregulated genes, Blue: downregulated genes, NC: No Change. Horizontal line denotes a p-value threshold of 0.01.
(C) Gene ontology enrichment analysis on the differentially expressed genes in the TDP43 model. The top five enriched pathways in upregulated and downregulated genes have been displayed.
(D) Venn diagram displaying the overlap between mis-spliced genes identified due to TDP43 mislocalization in the TDP43-GFP iPSC MNs (TDP43-misloc) and cortical neuronal nuclei displaying TDP43 depletion obtained from ALS/FTD patient tissue (GSE126542).
(E) Venn diagram displaying the overlap between mis-spliced genes identified due to TDP43 mislocalization in the TDP43-GFP iPSC MNs (TDP43-misloc) and TDP43 knockdown using siRNAs in healthy iPSC-MNs (GSE121569).
(F) RT-qPCR validating alternative splicing changes resulting from TDP43-GFP mislocalization in iPSC-MNs. MN samples were lysed at day 30, 12 days post-transduction with AAVs. Replicates were three independent differentiations of homozygous TDP43-GFP knock-in lines, (two of E5, one of E8). CE = cryptic exon, TRC = truncated, IR = intron retention. CACNA1E and KCNQ2 displayed alternate exon usage. The exons that were included in the NES samples have been indicated.
* indicated p < 0.05, ** indicates p < 0.01. *** indicates p < 0.001.
TDP43 Sustains its own Mislocalization
(A) Schematic depicting the knock-in of the nanobody (control or NES) into the human AAVS1 safe harbour locus of our E8 homozygous TDP43-GFP cell line. Nanobody expression was under the control of a doxycycline (Dox)-inducible promoter. Addition of Dox is expected to induce TDP43 mislocalization in the TDP43-GFP-NES MNs but not in the TDP43-GFP-Control (TDP43-GFP-CTRL) MNs.
(B) Immunofluorescent staining showing TDP43 localization in our TDP43-GFP-CTRL or TDP43-GFP-NES line in response to Dox. Mislocalization only occurs in the TDP43-GFP-NES line with Dox treatment, while all other conditions maintain nuclear TDP43. MNs were fixed and stained at day 35, 15 days post-Dox addition. Scale bar = 50 µm.
(C) TDP43 localization in the TDP43-GFP-NES cell line upon withdrawal of Dox. TDP43 fully mislocalizes following Dox treatment, which persists at 7 days post-withdrawal. After 14 days without Dox, a subset of cells show persistent TDP43 mislocalization in the cytoplasm (white arrow). Some cells show partial recovery of nuclear TDP43. All conditions (except the negative control) received a minimum of 72 hours in Dox prior to withdrawal. Scale bar = 20 µm.
(D) RT-qPCR depicting expression of the NES nanobody. Y-axis depicts ΔCt. Higher ΔCt Indicates lower expression. Ct values for each sample were normalized to RPL13, HPRT1 and GAPDH. At 14 days without Dox, nanobody transcript expression is reduced to levels similar to the No Dox condition though the difference was deemed significant due to the low variation in the replicates. The three colours indicate different biological replicates. 7D = 7 days post-Dox withdrawal, 14D = 14 days post-Dox withdrawal. +DOX = continuous Dox treatment, – DOX = never received Dox
(E) RT-qPCR showing mis-splicing of STMN2 at various stages of Dox-withdrawal. Despite some nuclear TDP43 recovery at day 14, canonical STMN2 transcript expression is reduced by 50%, with associated significant expression of the truncated STMN2 transcript. TRUNC = truncated.
(F) TDP43 localization in heterozygous TDP43-GFP knock-in lines. MNs were transduced at day 20 and fixed for immunostaining at day 35. TDP43-GFP (green) is almost exclusively cytoplasmic, while untagged TDP43 (magenta) is found in both the nucleus and cytoplasm. Approximately 50% of cells show near total nuclear clearance of endogenous TDP43. Scale bar = 50 µm.
** indicates p < 0.01, *** indicates p < 0.001.
(A) Confirmation of the three heterozygous and two homozygous TDP43-GFP knock-in clones using PCR-gel electrophoresis. The unedited parent healthy iPSCs served as a control.
(B) Representative images of TDP43-GFP homozygous clones E8 and E5 stained for MN markers neurofilament-M (NFM) and ISLET1 (ISL1) at day 40. Nuclei were stained with Hoechst 33342 Scale bar = 50 µm.
(C) RT-qPCR of unedited (healthy) iPSC-MNs and untransduced homozygous TDP43-GFP knock-in MNs at day 30, suggesting no adverse effects of GFP knock-in on TDP43 expression or alternative splicing. P-values were calculated using a one-tailed Student’s T-test. Error bars show SEM. None of the differences were significant.
n.s. = not significant
(A, B) Representative immunofluorescent images of decreased dendrite complexity in NES compared to control MNs. (A) Cells are from the E8 homozygous GFP knock-in cell line.
(B) Cells are from the E5 homozygous GFP knock-in cell line. Scale bar = 50 µm.
(C) Morphological analysis in unedited iPSC MNs treated with control or NES nanobodies. No significant effect is observed on dendrite morphology, soma swelling or apoptosis (CC3 intensity) a p-value threshold of 0.01. N=2 independent differentiations. At least 150 neurons were analyzed per replicate. CC3 = cleaved caspase-3
(D) Uncropped western blot (from Fig.1G) of soluble TDP43 in control (CTRL) and mislocalized (NES) MNs lysed at D42, 22 days post-transduction with AAVs. Clones represent independent differentiations of E5 and E8 homozygous TDP43-GFP knock-in lines.
(E) RT-qPCR showing abnormal splicing of UNC13A and STMN2 in day 40 homozygous TDP43-GFP MNs, 20 days post-transduction with NES or control nanobodies. Error bars show SEM. CE = cryptic exon, TRUNC = truncated.
n.s. = not significant, ** indicates p < 0.01,*** indicates p < 0.001.
UCSC Genome browser screen shots depicting cryptic exon inclusion (UNC13A, STMN2, ELAVL3) and intron retention (CELF5) resulting from TDP43 mislocalization. In all cases, there is at least one TDP43 binding site, identified by SH-SY5Y eCLIP data (GSE122648) shown as a horizontal black bar. Screenshots are from the homozygous E5 TDP43-GFP clone, at MN day 40, 20 days post-transduction with AAVs. Red arrows indicate the alternative splice event.
UCSC Genome browser shots depicting cryptic exon inclusion in CYFIP2, FEZ1, and IGSF21 resulting from TDP43 mislocalization. There are no TDP43 binding sites identified by SH-SY5Y eCLIP data (GSE122648). Screenshots are from the homozygous E5 TDP43-GFP clone, at MN day 40, 20 days post-transduction with AAVs. Red arrows indicate the alternative splice event.
(A) STMN2 transcripts containing the CE in the TDP43-GFP MNs expressing the NES nanobody (isoforms containing the variable length CE are shown in red, reference isoforms are show in dark blue at the top). Each isoform was assigned a unique number. Top five most abundant isoforms are marked by yellow arrows. Isoforms containing the extra 114 bp exon upstream of the CE are marked by purple arrows. (B) Quantification of the top five most abundant STMN2 isoforms in TDP43-GFP MNs expressing control or NES nanobody. Each dot represent data from an independent replicate. N=4.
RT-qPCR showing alternative splicing errors in unedited iPSC MNs, 10 days post-addition of shRNAs targeting TDP43, or controls. The chart showing successful TDP43 knockdown is highlighted in red. Error bars show SEM. CE = cryptic exon, TRC = truncated, IR = intron retention. N = 3 independent differentiations.
* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
(A) Immunofluorescent staining of day 22 TDP43-GFP-NES MNs showing TDP43 mislocalization at 48 hours post-treatment with 1 µg/ml doxycycline. Scale bar = 50 µm.
(B,C) RT-qPCRs showing alternative splicing errors in Dox-inducible TDP43-GFP-NES versus-CTRL cell lines at (B) 48 hours and (C) 20 days post-treatment with 1 µg/ml doxycycline. MNs were treated with doxycycline at day 20 of the differentiation process. CE = cryptic exon, TRC = truncated, IR = intron retention.
* indicates p < 0.05, ** indicates p < 0.01,*** indicates p < 0.001.
MicroRNA dysregulation in MNs due to TDP43 Mislocalization or Knockdown.
(A) Principal component analysis of microRNA (miR) counts for the TDP43-GFP-Control and TDP43-GFP-NES MNs.
(B) Volcano plot displaying miRs differentially expressed due to TDP43 mislocalization. Red: upregulated miRs, Blue: downregulated miRs, NC: No Change. Horizontal line denotes a p-value threshold of 0.01.
(C) Principal component analysis of miR counts for unedited healthy iPSC MNs transduced with TDP43 or control shRNAs.
(D) Volcano plot displaying miRs differentially expressed due to TDP43 knockdown. Blue: downregulated miRs, NC: No Change. Horizontal line denotes a p-value threshold of 0.01. No miRs were deemed significantly upregulated at the thresholds used.
(E, F) Gene-set enrichment analysis plots displaying overlap of differentially expressed miRs due to TDP43 mislocalization or knockdown. miRs differentially expressed in the TDP43 knockdown samples were ranked from most upregulated to the most downregulated. E: Gene set used for the GSEA include miRs downregulated due to TDP43 mislocalization, F: Gene set used for the GSEA include miRs upregulated due to TDP43 mislocalization.
Table of qPCR primers. AS = Alternative splicing.
