Extended Data Fig. 6: UNC13A cryptic splicing is associated with loss of nuclear TDP-43 in patients with FTD and motor neuron disease (MND).
From: TDP-43 represses cryptic exon inclusion in the FTD–ALS gene UNC13A
(a) Additional patients and control subjects used in the study (but not shown in Fig. 3), demonstrating UNC13A cryptic splicing. Scale bar equals 10 µm. (b) Quantification of UNC13A cryptic exon BaseScope™ in situ hybridization. Six non-overlapping Z-stack images from layer 2–3 of medial frontal pole were captured, per subject, using a 63X oil objective and flattened into a maximum intensity projection image. Puncta counts per image were derived using the “analyze particle” plugin in ImageJ. Each data point represents the number of UNC13A cryptic exon puncta in a single image. Cryptic exon quantity varies between patients but always exceeds the technical background of the assay, as observed in controls. (n = 6 non-overlapping Z-stack images; Linear mixed model, mean ± s.d.). (c) The design of the UNC13A e20/e21 BaseScope™ probe targeting canonical UNC13A transcript. Each “Z” binds to the transcript independently. Both “Z”s must be in close proximity for successful signal amplification, ensuring binding specificity. (d) Representative images showing expression of UNC13A mRNA in layer 2–3 neurons from the medial frontal pole using the probe shown in (b). UNC13A mRNA expression is restricted to neurons (arrows) and is decreased in cells exhibiting TDP-43 nuclear depletion. Arrowheads represent neurons with loss of nuclear TDP-43 and accompanying cytoplasmic inclusions, and arrows indicate neurons with normal nuclear TDP-43. Images are maximum intensity projections of a confocal image Z-stack. Scale bar equals 10 µm. (e) Quantification of UNC13A mRNA BaseScope™ in situ hybridization. UNC13A mRNA puncta were quantified as described in (b). Each data point represents the number of UNC13A mRNA puncta in a single image. This suggests some variability in UNC13A mRNA levels potentially attributable to technical or biological factors. More importantly, the control UNC13A mRNA levels suggest that failure to detect UNC13A cryptic exons in controls is not due to nonspecific RNA degradation. There is variability of UNC13A mRNA detected per sample but we observe a trend of reduced UNC13A mRNA in patient samples compared to controls. (Linear mixed model, mean ± s.d.). The final BaseScope™ experimental run was performed once involving all the cases and controls. Six non-overlapping images were captured from each individual, and representative images are shown. UNC13A probes were first optimized by testing them on 2 cases and 2 controls in 3 separate pilot experiments, showing similar findings.
