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. 2022 Dec 8;11(24):3972.
doi: 10.3390/cells11243972.

c-Abl Regulates the Pathological Deposition of TDP-43 via Tyrosine 43 Phosphorylation

Saebom Lee  1   2   3 , Hye Guk Ryu  4   5 , Sin Ho Kweon  1   2 , Hyerynn Kim  4 , Hyeonwoo Park  4 , Kyung-Ha Lee  5   6 , Sang-Min Jang  7 , Chan Hyun Na  1   2 , Sangjune Kim  4 , Han Seok Ko  1   2
Affiliations

c-Abl Regulates the Pathological Deposition of TDP-43 via Tyrosine 43 Phosphorylation

Saebom Lee et al. Cells. .

Abstract

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.

Keywords: TDP-43; amyotrophic lateral sclerosis; c-Abl; mislocalization; phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
c-Abl phosphorylates TDP-43 at tyrosine 43. (A) in vitro kinase assay with recombinant Kinase active form of c-Abl (c-Abl KA) and TDP-43. (B) Non-radioactive kinase assay adding cold ATP. c-Abl–phosphorylated TDP-43 indicated by asterisk. (C) MS/MS spectrum of the phosphorylated peptide fragment of c-Abl–phosphorylated TDP-43. (D) Co-immunoprecipitation experiments in SH-SY5Y cells co-transfected with EGFP-c-Abl and phospho-deficient mutant forms of TDP-43.
Figure 2
Figure 2
c-Abl interacts with TDP-43. (A) Co-immunoprecipitation experiments in SH-SY5Y cells co-transfected with Flag-c-Abl and EGFP-TDP-43. (B) The docking model for TDP-43 (red) interacting with c-Abl (cyan). Potential hydrogen bonds are shown as dashed lines.
Figure 3
Figure 3
c-Abl increases cytoplasmic distribution of endogenous TDP-43. (A) Immunocytochemical analyses of c-Abl and TDP-43. GFP-c-Abl KA (green) or GFP-c-Abl KD and TDP-43-TdTomato (red) were co-expressed in SH-SY5Y cells. (B) Quantification of the percentage of cells expressing cytoplasmic TDP-43 (n = 4–12). (C) Expression of kinase active (KA) form of c-Abl increases the accumulation of TDP-43 in the cytoplasm, but not in expression of kinase dead (KD) form of c-Abl. (D) Quantification of the levels of cytoplasmic TDP-43 in the c-Abl KA and KD–expressing cells (n = 3). Statistical significance was determined by using one-way ANOVA with Tukey’s correction; * p < 0.05, ** p < 0.01, n.s. = non-significance.
Figure 4
Figure 4
Y43 residue of TDP-43 associates with the TDP-43 pathology. (A) Immunostaining with G3BP1 antibody that can detect cytoplasmic stress granules in SH-SY5Y cells transfected with GFP-WT TDP-43, GFP-phospho-mimic forms of Y43E and Y155E TDP-43. (B) Quantification of the percentage of cells with cytoplasmic stress granules (n = 3). Statistical significance was determined by using one-way ANOVA with Tukey’s correction; * p < 0.05, n.s. = non-significance. (C) Increased localization of TDP-43 at neurite granules by c-Abl KA co-expression in cultured cortical neurons. TDP-43 TdTomato and EGFP-c-Abl were expressed in the cultured cortical neurons and followed by immunocytochemistry. (D) Phospho-mimic form of TDP-43 Y43E showed increased neurite granule expression than TDP-43 WT in primary cultured cortical neurons. EGFP-TDP-43 WT and Y43E were expressed alone in the cultured cortical neurons and followed by immunocytochemical alayses.
Figure 5
Figure 5
TDP-43 Y43E increases aggregates and neuronal cell death in primary cortical neurons. (A) RIPA/urea-fractionated cell lysates with RIPA (Soluble) and urea (Insoluble) buffers from c-Abl WT and c-Abl KO neurons were examined using TDP-43 antibody (B) Urea-fractionated cell lysates from c-Abl WT and c-Abl KO were collected and subjected to dot blotting by TDP-43 antibody. (C,D) TUNEL staining of the cortical neurons shows neurotoxicity of Lenti-viral induced overexpression of indicated TDP-43 plasmids. The percentage of TUNEL-positive neurons showing apoptotic nuclei is quantified and shown in (D). Statistical significance was determined by using one-way ANOVA with Tukey’s correction; * p < 0.05, ** p < 0.01, *** p < 0.001.
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