TDP-43 interacts with mitochondrial proteins critical for mitophagy and mitochondrial dynamics

Abstract

Transactive response DNA-binding protein of 43 kDa (TDP-43) functions as a heterogeneous nuclear ribonucleoprotein and is the major pathological protein in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis/motor neuron disease (ALS/MND). TDP-43 pathology may also be present as a comorbidity in approximately 20-50% of sporadic Alzheimer's disease cases. In a mouse model of MND, full-length TDP-43 increases association with the mitochondria and blocking the TDP-43/mitochondria interaction ameliorates motor dysfunction. Utilizing a proteomics screen, several mitochondrial TDP-43-interacting partners were identified, including voltage-gated anion channel 1 (VDAC1) and prohibitin 2 (PHB2), a crucial mitophagy receptor. Overexpression of TDP-43 led to an increase in PHB2 whereas TDP-43 knockdown reduced PHB2 expression in cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inducer of mitophagy. These results suggest that TDP-43 expression contributes to metabolism and mitochondrial function however we show no change in bioenergetics when TDP-43 is overexpressed and knocked down in HEK293T cells. Furthermore, the fusion protein mitofusin 2 (MFN2) interacts in complex with TDP-43 and selective expression of human TDP-43 in the hippocampus and cortex induced an age-dependent change in Mfn2 expression. Mitochondria morphology is altered in 9-month-old mice selectively expressing TDP-43 in an APP/PS1 background compared with APP/PS1 littermates. We further confirmed TDP-43 localization to the mitochondria using immunogold labeled TDP-43 transmission electron microscopy (TEM) and mitochondrial isolation methods There was no increase in full-length TDP-43 localized to the mitochondria in APP/PS1 mice compared to wild-type (littermates); however, using C- and N-terminal-specific TDP-43 antibodies, the N-terminal (27 kDa, N27) and C-terminal (30 kDa, C30) fragments of TDP-43 are greatly enriched in mitochondrial fractions. In addition, when the mitochondrial peptidase (PMPCA) is overexpressed there is an increase in the N-terminal fragment (N27). These results suggest that TDP-43 processing may contribute to metabolism and mitochondrial function.

Keywords: APP/PS1; MFN2; Mitochondria; Mitophagy; PHB2; PMPCA; TDP-43.

Figure 1. TDP-43 expression alters PHB2 expression and induces LC3-II

A) Immunoprecipitation of TDP-43 and immunoblot (IB) with anti-PHB2. mIgG indicates mouse IgG control, rIgG indicates rabbit IgG control. B) Immunoprecipitation of PHB2 and TDP-43 and immunoblot for TDP-43. C) Western blot for TDP-43, PHB2, LC3B, COXIV and Tubulin from lysed HEK293T cells transfected with siNC (negative control), siTDP-43, vector (PLX), or v5 tagged TDP-43 (TDP-43v5) and treated with either 10μM CCCP (indicated by + above lane) or DMSO as a control (indicated by -) for 12 hours. D-E) Quantitation of TDP- 43/Tubulin from Western blot above. F-G) Quantitation of PHB2/Tubulin from Western blot above. H-I) Quantitation of LC3-II/tubulin from Western blot above. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA with Tukey correction for Type I error. Error bars represent standard deviation.

Figure 2. Selective expression of TDP-43 induces an age-dependent increase in Mitofusin-2

A) Transmission electron microscopy of hippocampal neurons in 9-month old APP/PS1 and Camk2a/hTDP- 43/APP/PS1 at 20,000×. B) Western blot for MFN2, OPA1, phospho-DRP1 (pDRP1) (Ser637), COXIV and Tubulin from lysate of cortex extracted from 4-month-old non-transgenic (WT), APP/PS1, Camk2a/hTDP-43, and Camk2a/hTDP-43/APP/PS1 mice. C & D) Quantitation of 4-month-old MFN2 and pDRP1 expression over Tubulin normalized to wild-type. E) Western blot for MFN2, OPA1, pDRP1(Ser637), and Tubulin from lysate of cortex extracted from 9-month-old non-transgenic (WT), APP/PS1, and Camk2a/hTDP-43 mice. F & G) Quantitation of 9-month-old MFN2 and pDRP1 expression over Tubulin normalized to wild-type.

Figure 3. Mitochondrial fraction enhanced TDP-43 N- and C- terminal fragments

Transmission electron micrograph of immunogold labeled TDP-43 in the hippocampus of 8-month-old A) wild-type and B) APP/PS1 mice. TDP-43 localizes around the mitochondria in wild-type and APP/PS1 mice; 19,500× (left) and 53,000× (right) magnification. C) Mitochondrial fraction (Percoll gradient) from the cortex/hippocampus of 9-month-old wild-type and APP/PS1 mice, immunoblotted for TDP-43 N-terminal (N-t), C-terminal (C-t), tubulin, COXIV, Histone H3, and APP (6E10) D) Quantification of mitochondrial full-length TDP-43 normalized to COXIV. E) Quantification of N-terminal TDP-43 (27kDa) normalized to COXIV in mitochondrial fractions (band indicated by N27 in C). F) Quantification of C-terminal TDP-43 (30kDa) normalized to COXIV in mitochondrial fractions (band indicated by C30 in C). G) Quantification of 35kDa TDP-43 (N-terminal) from whole cell lysate normalized to Tubulin. H) Quantification of 25kDa TDP- 43 (C-terminal) from whole cell lysate normalized to Tubulin. I) Quantification of histone H3 normalized to Tubulin from whole cell lysates. T, total lysate; M, mitochondrial fraction; *p<0.05, **p<0.01. Unpaired t test, two-tailed. Error bars represent standard deviation. J) Domain structure of TDP-43 displaying antibody epitopes for TDP-43 N-terminal (1-30) and C-terminal (401-414) and the various forms of TDP- 43 detected by each from cortex/hippocampus mitochondrial fractions and total lysates.