Cytoplasmic TDP43 Binds microRNAs: New Disease Targets in Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, and incurable neurodegenerative disease. Recent studies suggest that dysregulation of gene expression by microRNAs (miRNAs) may play an important role in ALS pathogenesis. The reversible nature of this dysregulation makes miRNAs attractive pharmacological targets and a potential therapeutic avenue. Under physiological conditions, miRNA biogenesis, which begins in the nucleus and includes further maturation in the cytoplasm, involves trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43). However, TDP43 mutations or stress trigger TDP43 mislocalization and inclusion formation, a hallmark of most ALS cases, that may lead to aberrant protein/miRNA interactions in the cytoplasm. Herein, we demonstrated that TDP43 exhibits differential binding affinity for select miRNAs, which prompted us to profile miRNAs that preferentially bind cytoplasmic TDP43. Using cellular models expressing TDP43 variants and miRNA profiling analyses, we identified differential levels of 65 cytoplasmic TDP43-associated miRNAs. Of these, approximately 30% exhibited levels that differed by more than 3-fold in the cytoplasmic TDP43 models relative to our control model. The hits included both novel miRNAs and miRNAs previously associated with ALS that potentially regulate several predicted genes and pathways that may be important for pathogenesis. Accordingly, these findings highlight specific miRNAs that may shed light on relevant disease pathways and could represent potential biomarkers and reversible treatment targets for ALS.

Keywords: amyotrophic lateral sclerosis; cytoplasmic aggregates; microRNAs; profiling; trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43).

Figure 1

Trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43) binding specificity and apparent binding affinities for select microRNAs (miRNAs). (A) A native gel mobility shift assay was used to evaluate the association between TDP43 and previously associated miRNAs: miR-132, miR-143, and miR-574. Trace amounts of ³²P-radiolabeled RNA (5 nM) were incubated with increasing concentrations of TDP43 (0, 0.05, 0.125, 0.25, 0.5, and 1.25 mM) and analyzed on a 12.5% native PAGE gel. A shift in band size was observed only for miR-574 (right). (B) Apparent binding affinities (EC50s) of TDP43 for select miRNAs with varying numbers of UG repeats were determined using a titration of biotinylated miRNAs in a colorimetric ELISA. EC50s are proportional to the number of UG repeats in each miRNA (miR-574, nine repeats > miR-652, three repeats > miR-204, two repeats).

Figure 2

Doxycycline-inducible stable cell lines expressing TDP43 variants. (A) Representative images of control (eGFP-His), wild-type (WT)-TDP43-eGFP-His (WT-TDP43), and ΔNLS-TDP43-eGFP-His (ΔNLS-TDP43) stable HeLa cell lines depict primarily nuclear expression for WT-TDP43 and predominantly cytoplasmic expression for ΔNLS-TDP43. Scale bar = 50 μm. (B) Immunoblotting of whole-cell lysates and cytoplasmic and nuclear fractions for α-tubulin (cytoplasmic marker; top panel), H2B (nuclear marker; middle panel), and eGFP (a marker for expression of the eGFP-His-tagged doxycycline-inducible plasmids) following subcellular fractionation of the control (C), WT-TDP43-eGFP-His (WT), and ΔNLS-TDP43-eGFP-His (Δ) lines. eGFP immunoblotting demonstrates that eGFP-His (lower band) is expressed in the whole-cell lysates and cytoplasmic fraction of the control cell line, while eGFP-His-tagged TDP43 (upper band) is present in the whole-cell lysates and cytoplasmic fraction of the WT-TDP43-eGFP-His and ΔNLS-TDP43-eGFP-His cell lines, although only marginal expression seen in the WT-TDP43-eGFP-His cell line. (C) TDP43 immunoblotting following pull-down of the His-tagged proteins using Ni²⁺ beads. ΔNLS-TDP43-eGFP-His is detected mainly in the cytoplasmic fraction, with some expression also seen in the nuclear fraction, while WT-TDP43-eGFP-His is more highly abundant in the nuclear fraction, though modest expression is also seen in the cytoplasmic fraction. The slightly higher bands in both TDP43 lines represent the TDP43-eGFP-His fusion proteins, while the lower bands represent endogenous TDP43. In the cytoplasmic fraction, ΔNLS-TDP43-eGFP-His also exhibits a third band (lowest) that likely reflects a proteolytic cleavage product of TDP43. C = eGFP-His; WT = WT-TDP43-eGFP-His; Δ = ΔNLS-TDP43-eGFP-His; B = Ni²⁺ bead only control; M = marker/ladder.

Figure 3

Enriched miRNAs associated with cyTDP43. miRNAs in the cytoplasmic fractions of the control (eGFP-His), WT-TDP43-eGFP-His (WT-TDP43), and ΔNLS-TDP43-eGFP-His (ΔNLS-TDP43) stable cell lines were profiled using NanoString technology. Each individual miRNA was normalized to the top 100 miRNAs and fold-change was determined relative to the control line; n = 4 per line. (A) Heat map of the fold-change of the top enriched miRNAs. Red represents positive values (increased enrichment), while green represents negative values (decreased enrichment) for the WT-TDP43 (left) and ΔNLS-TDP43 (right) lines relative to the control (eGFP-His) line. (B,C) Fold change of top enriched miRNAs in the cytoplasmic fraction of the WT-TDP43 (B) and ΔNLS-TDP43 (C).

Figure 4

Confirmation of select enriched miRNAs. Levels of select miRNAs from the NanoString profiling results in cytoplasmic fractions of the control (eGFP-His), WT-TDP43-eGFP-His (WT-TDP43), and ΔNLS-TDP43-eGFP-His (ΔNLS-TDP43) lines were confirmed using quantitative real-time PCR (qPCR). Results were normalized to miR-92a (ΔCT) and then relative to the control group (ΔΔCT) and expressed as mean fold-change ± standard error of the mean (SEM; n = 4 per line). (A) miR-204-5p was significantly upregulated relative to control in both TDP43 lines in a similar proportion to the NanoString results (*p < 0.05). (B) miR-129-5p levels in both TDP43 lines were upregulated at fold-changes equivalent to the NanoString results, but did not reach significance relative to the control line. (C) Fold-change comparisons between the qPCR and NanoString profiling results.

Figure 5

miRNA pathway analysis of the top enriched miRNAs. Predicted gene targets and associated KEGG pathways following pathway analysis of the top ΔNLS-TDP43 (A) and WT-TDP43 (B) miRNAs using mirPath v.3. Higher intensity of red shading reflects increasing significance values, while the size of the nodes represents the number of altered miRNAs within the pathway. Lists of the predicted pathways represented among the ΔNLS-TDP43 and WT-TDP43 enriched miRNAs are also detailed in Supplementary Tables S1, S2.