Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS-FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.

Figure 1.. TDP-43 depletion or genome editing in a human neuronal cell line identifies significant reduction in stathmin-2 expression levels.

(a) Quantitative real-time PCR analysis confirming siRNA-mediated reduction of TDP-43 mRNA levels in SH-SY5Y cells. Expression of TRFC and GAPDH mRNAs were used as endogenous controls. Cells were treated with siControl (white bar, black dots, mean=1) or siTDP-43 (gray bar, red dots, mean=0.19) for 96 hours in three biologically independent experiments (two-tailed t-test, P=0.0012, n=3), error bars represent SEM, **P<0.01. (b) Volcano-plot showing differentially expressed genes in SH-SY5Y cells depleted of TDP-43 by siRNA treatment. Genes with significant changes in mRNA levels are represented by red dots (n=3 biologically independent experiments, fold change >1.5 and FDR<0.05) by DESeq2 . Increased red dot size represents increased statistical significance (measured by FDR<0.05). RNA-seq analysis identified 518 misregulated genes and confirmed 4 fold reduction in TDP-43 mRNA levels. Stathmin-2 mRNA showed the strongest reduction (6.5 fold) upon TDP-43 depletion. Up and down regulated genes’ counts are indicated. Expression values were determined as transcripts per kilobase per million mapped reads (TPM). (c) Quantitative real-time PCR analysis confirming reduction of stathmin-2 mRNA expression levels (mean=0.13) in SH-SY5Y cells treated with siRNA targeting TDP-43 compared to cells treated with siControl (mean = 1) for 96 hours in three biologically independent experiments (two-tailed t-test, P=0.0005, n=3), error bars represent SEM. ^***P<0.001 (d) Immunoblotting of TDP-43 and stathmin-2 in SH-SY5Y cells treated with siControl or siTDP43 for 96 hours. α-tubulin served as a loading control. Three biological replicates are shown. (e) Representative Immunofluorescence of TDP-43 (green) and lamin-B (red) in SH-SY5Y lines expressing wild type or mutant TDP-43 by genome-editing. Genotypes are indicated, experiment was reproduced 3 times independently with similar results. For unprocessed blots, see Supplementary Fig. 10. (f) Volcano plot depicting 950 differentially expressed genes identified by genome-wide RNA-seq. Significant changes in mRNA levels between SH-SY5Y^WT and SH-SY5Y^N352S/N352S lines are represented by red dots (n=2 biologically independent experiments, fold change > 1.5, FDR<0.05) by DESeq2 . Increased red dot size represents increased statistical significance (measured by FDR<0.05). Expression values were calculated as transcripts per kilobase per million mapped reads (TPM). (g) Quantitative real-time PCR analysis confirming significant reduction in stathmin-2 mRNA levels between isogenic SH-SY5Y^WT/WT (white bar, black dots, mean=1) and SH-SY5Y^N352S/N352S (black bar, red dots, mean=0.58) lines. Expression of TRFC and GAPDH mRNAs were used as endogenous controls (two-tailed t-test, P=0.004, n=3 independent biological experiments), error bars represent SEM. ^**P<0.01. (h) Expression changes of 42 overlapping genes from figures 1b and 1f are plotted.

Figure 2.. Reduced stathmin-2 levels in human neurons produced by direct conversion from ALS patient fibroblasts expressing mutant TDP-43.

(a) Summary of the direct conversion strategy adopted from Xue et al. 2013 . (b) Induced neurons (iNeurons) converted from fibroblasts of control and ALS patient. Immunofluorescence staining at day 20 of differentiation revealed partial cytoplasmic mislocalization of TDP-43 (green) in ALS iNeurons. Neuron-specific Class III β-tubulin (Tuj1) (red) was used as a neuronal marker, cell nuclei are visualized by DAPI staining (blue). Experiment was repeated independently with similar results from 3 control individuals and 3 familial ALS donor lines (with mutant TDP-43). (c) qPCR analysis of stathmin-2 mRNA levels in iNeurons from control (grey bars, black dots) and familial ALS patients with different TDP-43 mutations (white bars, turquoise dots). Plotted are 2 biologically independent experiments per each individual from an extended family that included 4 carriers heterozygous for the ALS-linked mutant TDP-43^N352S (n=4, mean=0.3) and 4 individuals without the mutation (n=4, mean=1). Two-tailed t-test, P = 0.008. Also plotted are three biologically independent experiments of three additional ALS patients (non-family members) carriers of the indicated mutations. (d) Summary of stathmin-2 expression level measured by qPCR in iNeurons from controls (n=4, gray, mean = 1) and familial ALS patients (n=7, white, mean=0.36). P=0.0005, two-tailed t-test, SEM. ^**P<0.01^***, ^***P<0.001.

Figure 3.. TDP-43 regulates stathmin-2 mRNA levels by repressing premature polyadenylation.

(a) RNA-seq reads mapped to the genomic region of stathmin-2 reveal incorporation of a new exon, originated from intron 1, into the mature stathmin-2 mRNA. Red arrows indicate the intronic region of aberrant splicing (exon 2a) in SH-SY5Y cells upon TDP-43 depletion (upper panels) or expression of mutant TDP-43 (lower panels). Experiment was repeated independently three times (TDP-43 depletion) or twice (expression of mutant TDP-43) with similar results. (b) Representative RT-PCR (left, experiment was repeated 3 times independently with similar results) and qPCR (right, n=3 biologically independent experiments) analyses confirmed expression of the new spliced mRNA isoform containing exon 2a upon TDP-43 depletion (mean=8.7, P=0.00078) or mutation (mean=3.8, P=0.0001). The location of primers in exons 1 and 2a is shown. RT-PCR of the CENP-A transcript was used as loading control for RT-PCR, TFRC and GAPDH were used as qPCR normalizers (***p<0.001, two-tailed t-test, SEM). For uncropped gel images, see Supplementary Fig. 10. (c) Schematic representation of stathmin-2 pre-mRNA (upper panel) and alternative RNA isoforms (lower panels) in normal cells (i) or cells with TDP-43 deficiency (ii). Constitutive exons are represented by black boxes, exon 2a is in red and the thin red box represents the newly acquired 3’UTR. Light grey boxes represent 3’ or 5’ UTRs of normal stathmin-2 mRNA. (d) Sequence of exon 2a is shown in red, including the embedded in-frame UAG codon generating a premature stop codon. Highlighted are potential TDP-43 binding sites located 127 upstream of the alternative polyadenylation signal. (e) 3’ end sequencing by reverse transcription using oligo(dT)-VN primers confirmed exon 2a as the terminal exon of short stathmin-2 mRNA. (f) Genome browser track obtained from cross-linking and immuno-precipitation (iCLIP) for TDP-43 in human SH-SY5Y cells , revealed TDP-43 physical binding to exon 2a, located in intron 1 of stathmin-2 pre-mRNA.

Figure 4.. Abnormal stathmin-2 mRNA processing is a disease hallmark in affected spinal motor neurons of sporadic ALS patients.

(a) RNA sequencing of control and sporadic ALS laser-captured spinal motor neurons reveals robust signature of exon 2a incorporation in stathmin-2 mRNA in sporadic ALS samples but not in non-ALS aged controls. Lower diagram shows the genomic region of stathmin-2 and mapped RNA reads of exon 2a are indicated by a red arrow. Data analyzed from from Krach et al., 2018 . (b) Stathmin-2 mRNA expression is suppressed in spinal motor neurons of sporadic ALS patients (gray bar, n=13, mean = 423 TPM, P=0.03) relative to non-ALS aged controls (white bar, n=7, mean=971 TPM, P=0.019). Two-tailed t-test, SEM. RNA sequencing data was analyzed from Krach et al., 2018 . Stathmin-1 mRNA expression is shown to the right (control mean=25, ALS patients mean = 44.5). Transcripts per million counts (TPM) are represented, two-tailed t-test. P=0.02. *P<0.05, SEM. (c-d) RT-PCR using primers targeted to exon 1 and exon 2a confirms expression of truncated stathmin-2 isoform in (c) thoracic spinal cord or (d) motor cortex from sporadic and C9orf72 ALS patients, but not in spinal cords of mutant SOD1 carriers. For uncropped gel images, see Supplementary Fig. 10 (e-f) Lumbar spinal cord and motor cortex sections isolated from control individuals and sporadic ALS patients were hybridized with locked nucleic acid (LNA) probes targeting intron one of stathmin-2 pre-mRNA (for the truncated RNA) or exon 5 of stathmin-2 pre-mRNA. Signal is in blue, counterstain is nuclear fast red. (g) Table summarizing human samples with expression of stathmin-2 truncated RNA, identified by RT-PCR or in-situ hybridization. Data from supplementary Fig. 7 is included.

Figure 5:. Impaired axonal regeneration upon TDP-43 loss in human iPSC-derived motor neurons is alleviated by stathmin-2 restoration.

(a) Quantitative real-time PCR analysis confirming ASO-mediated reduction of TDP-43 mRNA (gray, dose-dependent mean values are 0.71, 0.6, 0.43, respectively) or stathmin-2 mRNA (red, dose-dependent mean values are 0.67, 0.31, 0.09, respectively) levels in human iPSC-derived motor neurons. Control bar (white, mean=1) represents 3 biological replicates, expression of TRFC mRNA was used as endogenous control. Error bars represent SEM. (b) RT-PCR analysis confirmed altered splicing and ligation of exon 1 and exon 2a upon treatment with ASOs targeting TDP-43, in iPSC-derived motor neurons. Three ASO concentrations were tested and a dose dependent effect on expression of stathmin-2 short RNA is shown. Experiment was reproduced five times independently with similar results. For uncropped gel images, see Supplementary Fig. 10. (c) Quantitative real-time PCR analysis confirming ASO-mediated reduction of stathmin-2 mRNA (red, dose-dependent mean values are 0.23, 0.12, 0.07, respectively) or TDP-43 mRNA (gray, dose-dependent mean values are 0.99, 0.95, 1.04, respectively) levels in human iPSC-derived motor neurons. Control bar (mean=1) represents 3 biological replicates, expression of TRFC mRNA was used as endogenous control. Error bars represent SEM. (d) Immunoblotting of TDP-43 and Stathmin-2 in iPSC-derived motor neurons treated with mouse Malat-1 ASO as control (n=2 biologically independent experiments), TDP-43 ASO (n=2 biologically independent experiments) or stathmin-2 ASO (n=2 biologically independent experiments) for 20 days. α-Tubulin served as a loading control. Stathmin-2 protein was profoundly suppressed following TDP-43 ASO or stathmin-2 ASO treatments. Experiment was repeated independently five times with similar results. For uncropped blots, see Supplementary Fig. 10. (e) Schematic illustration of motor neurons in microfluidic chambers before and after axotomy. Motor neuron precursors were plated at the somatic/proximal compartment and axons reached into the distal/axonal compartment during a maturation period of another eight days. One dose of ASOs was added every 12 days to the somatic compartment. After 20 days of culturing with ASOs, aspiration-based axotomy was performed at the distal compartment of each chamber leading to a complete removal of axons in that compartment. Recovery was allowed for 24 hours. (f) Timeline of iPSC-derived motor neuron maturation, ASO treatment and axotomy is shown. (g,i,k,m,o) Representative confocal immunofluorescence images of microgrooves and distal compartments, 24 hours post axotomy. Insets show high-magnification of regenerating axons’ growth cones. Axonal regeneration and growth cones are observed by immunofluorescence of stathmin-2 (green) and NF-H (red) in the terminals of motor neurons treated with control ASOs (g,h) but not in motor neurons treated with stathmin-2 (i,j) or TDP-43 (k,l) ASOs. (m,n,o,p) iPSCs-derived motor neurons treated with ASOs against stathmin-2 (m,n) or TDP43 (o,p) for 20 days and transduced with stathmin-2 expressing lentivirus for the last 96 hours showed axonal regeneration with growth cones in all axonal terminals. (h,j,l,n,p) Quantifications of 300 axons per condition are shown.