Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia

Abstract

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

Keywords: Dementia; Neuroscience.

Figure 1. Truncated STMN2 RNA is generated with loss of nuclear TDP-43.

Schematic of TDP-43–regulated STMN2 splicing. When TDP-43 is present and functional, it binds to GU-rich sequences in the first intron of the STMN2 transcript and allows normal splicing of intron 1. Reduced TDP-43 binding to STMN2 RNA, from TDP-43 aggregation or depletion from the nucleus, leads to mis-splicing of STMN2 RNA, resulting in the inclusion of a novel exon encoded within intron 1 (termed exon 2a) and containing an alternative or cryptic polyadenylation site. The production of this alternative variant of STMN2, which lacks exons 2 through 5 (referred to as truncated STMN2), is at the expense of full-length STMN2, which is reduced upon TDP-43 downregulation.

Figure 2. Truncated STMN2 RNA is elevated in iPSC-derived neurons with reduced levels of TDP-43.

(A–C) iPSC-derived cortical neurons constitutively expressing CRISPR inactivation machinery (data set labeled “a”) were transduced with a lentivirus expressing a sgRNA against TARDBP (TDP-43 KD) or a control sgRNA (Controls) and subjected to analysis by qRT-PCR (n = 3 TDP-43 KD, n = 3 controls) and/or RNA-Seq (n = 3 TDP-43 KD, n = 4 controls). The graphs show a decrease in TARDBP transcripts (A), a reduction of the full-length STMN2 transcript (B), and an increase in the truncated STMN2 transcript (C) upon TDP-43 depletion. (D) Detection of STMN2 cryptic exon 2a inclusion expressed as PSI by RNA-Seq in various iPSC-derived neurons: iPSC-derived cortical neurons (data set labeled “a”) described above and performed in this study; iPSC-derived motor neurons treated with TARDBP siRNA (TDP-43 KD) or control siRNA (controls) from Klim et al. (data set labeled “b”) (n = 6 TDP-43 KD, n = 11 controls) (7); iPSC-derived motor neurons from patients with TARDBP mutations (TDP-43 mut.) or controls from 2 independent groups in Edinburgh (data set labeled “c”) (n = 7 TDP-43 KD, n = 4 controls) and Oxford (data set labeled “d”) (n = 10 TDP-43 KD, n = 4 controls) (72). Data are presented as the mean ± SEM and were normalized to the control groups in A and B (set to 100%). *P < 0.05, ***P < 0.005, and ****P < 0.001, by Student’s t test (A–C) or 1-way ANOVA (D). (E) Representative images of iPSC-derived motor neurons from patients with TARDBP mutations and control subjects from a group in Oxford (data set labeled “d” in Figure 1D) showed similar nuclear-cytoplasmic distribution of TDP-43 (green) on day 30 of differentiation (DAPI in blue, choline acetyltransferase [ChAT] in white). Scale bar: 10 μm. All panels of Figure 2E are reshown in Supplemental Figure 3A. Additional data associated with this figure can also be found in Supplemental Figure 3. See also the complete unedited blots in the supplemental material.

Figure 3. Truncated STMN2 RNA is detected in bulk RNA-Seq from FTLD/ALS tissues with TDP-43 pathology.

(A) The NYGC ALS Consortium RNA-Seq data set was analyzed for the presence of truncated STMN2 transcripts, which were identified by RNA-Seq reads spanning the exon 1–exon 2a splice junction. (B–E) Bar graphs represent the proportion of individuals (percentage) with at least 2 reads spanning the exon 1–exon 2a junction. The number of individuals for the samples of the indicated tissues/diseases is indicated. (B) Truncated STMN2 was detected in the frontal cortex of patients with FTLD-TDP but not in control subjects or patients with FTLD-FUS or FTLD-tau. (C) In the lumbar spinal cord of patients with ALS, truncated STMN2 RNA was only detected in ALS-TDP samples but not in ALS-SOD1 or control samples. (D) Among FTLD-TDP tissues, truncated STMN2 was only detected in those known to have TDP-43 pathology (frontal and temporal cortex), whereas (E) within ALS-TDP tissues, truncated STMN2 RNA was seen in motor cortex and spinal cord tissues but not in regions without TDP-43 pathology.

Figure 4. Truncated STMN2 is elevated in FTLD-TDP and associates with higher p–TDP-43 burden and earlier age of FTD onset.

(A and B) Truncated and full-length STMN2 levels were measured in RNA extracted from frontal cortex of control subjects and FTLD-TDP and PSP patients, using the NanoString PlexSet platform. (A) A significant accumulation of truncated STMN2 RNA was detected in patients with FTLD-TDP but not in controls (see also Supplemental Table 2) or patients with PSP (see also Table 2). (B) Full-length STMN2 levels were significantly decreased in FTLD-TDP and PSP patients (see also Supplemental Table 3). Data are presented as the median with a 95% CI, and P values were determined using linear regression models that were adjusted for age at death, sex, and RIN. *P < 0.05, ***P < 0.005, ****P < 0.001. (C and D) In our FTLD-TDP cohort (n = 238), significant correlations were observed between truncated STMN2 RNA levels and (C) a higher burden of p–TDP-43 and (D) an earlier age of disease onset. See Table 3 for the correlation coefficients.